Gene DNA Methylation Research Articles

P129 DNA methylation of the tumour necrosis factor gene as a biomarker of diagnosis in early rheumatoid arthritis patients

Abstract Background/Aims Rheumatoid arthritis (RA) is an autoimmune inflammatory condition that affects the synovial joints. RA exists on a disease continuum where immunological changes occur before the onset of clinical symptoms. Changes in DNA methylation status of genes are thought to be early triggers of the progression from health to disease. Tumour necrosis factor (TNF) is a pro-inflammatory cytokine that contributes to RA. Recently it has been shown that the loss of DNA methylation in the promoter region of the TNF gene is related to early RA. This study aims to establish the predictive value of the levels of DNA methylation of the TNF gene as a biomarker for diagnosis of RA. Methods Blood samples from a retrospective cohort of patients with symptoms of inflammatory arthritis between 2010 and 2019 at the Leeds Early Arthritis Clinic were selected. Data obtained include clinical symptoms of disease progression over 2 years to establish a diagnosis. DNA methylation levels of the TNF gene were measured by quantitative polymerase chain reaction (qPCR) and analysed using binary logistic regression with forward (LR) variable selection for added value as a diagnostic biomarker. Results For the 284 patients with a mean age of 53.8, 202 (71.7%) were female and 82 (28.9%) were male. From N = 284, 190 (66.9%) were diagnosed with RA and 94 (33.1%) with another conditions. The predictive accuracy of the regression model was increased by 3.5% from 85.8% to 89.3% when TNF DNA-methylation levels was added to demographic and clinical variables. The AUC of the model with TNF was 0.940 compared to 0.911 without TNF DNA-methylation levels. The sensitivity and specificity of the prediction with DNA methylation data were 94.6% and 77.9% respectively. This TNF model has a positive predictive value of 90.2% and a negative predictive value of 87.0%. Conclusion DNA methylation of the TNF gene is an important biomarker for RA diagnosis. This study showed that including levels of TNF DNA-methylation improves the accuracy of diagnosing RA. Disclosure M.C. Liu: None. H. Ng: None. F. Ponchel: None.

IGEM as a human iPS cell-based global epigenetic modulation detection assay provides throughput characterization of chemicals affecting DNA methylation

Chemical-induced dysregulation of DNA methylation during the fetal period is known to contribute to developmental disorders or increase the risk of certain diseases later in life. In this study, we developed an iGEM (iPS cell-based global epigenetic modulation) detection assay using human induced pluripotent stem (hiPS) cells that express a fluorescently labeled methyl-CpG-binding domain (MBD), which enables a high-throughput screening of epigenetic teratogens/mutagens. 135 chemicals with known cardiotoxicity and carcinogenicity were categorized according to the MBD signal intensity, which reflects the degree of nuclear spatial distribution/concentration of DNA methylation. Further biological characterization through machine-learning analysis that integrated genome-wide DNA methylation, gene expression profiling, and knowledge-based pathway analysis revealed that chemicals with hyperactive MBD signals strongly associated their effects on DNA methylation and expression of genes involved in cell cycle and development. These results demonstrated that our MBD-based integrated analytical system is a powerful framework for detecting epigenetic compounds and providing mechanism insights of pharmaceutical development for sustainable human health.

3D genome mapping identifies subgroup-specific chromosome conformations and tumor-dependency genes in ependymoma

Ependymoma is a tumor of the brain or spinal cord. The two most common and aggressive molecular groups of ependymoma are the supratentorial ZFTA-fusion associated and the posterior fossa ependymoma group A. In both groups, tumors occur mainly in young children and frequently recur after treatment. Although molecular mechanisms underlying these diseases have recently been uncovered, they remain difficult to target and innovative therapeutic approaches are urgently needed. Here, we use genome-wide chromosome conformation capture (Hi-C), complemented with CTCF and H3K27ac ChIP-seq, as well as gene expression and DNA methylation analysis in primary and relapsed ependymoma tumors, to identify chromosomal conformations and regulatory mechanisms associated with aberrant gene expression. In particular, we observe the formation of new topologically associating domains (‘neo-TADs’) caused by structural variants, group-specific 3D chromatin loops, and the replacement of CTCF insulators by DNA hyper-methylation. Through inhibition experiments, we validate that genes implicated by these 3D genome conformations are essential for the survival of patient-derived ependymoma models in a group-specific manner. Thus, this study extends our ability to reveal tumor-dependency genes by 3D genome conformations even in tumors that lack targetable genetic alterations.

Common factors among three types of cells aged in mice.

The greatest risk factor for the formation of numerous significant chronic disorders is aging. Understanding the core molecular underpinnings of aging can help to slow down the inevitable process. Systematic study of gene expression or DNA methylation data is possible at the transcriptomics and epigenetics levels. DNA methylation and gene expression are both affected by aging. Gene expression is an important element in the aging of Homo sapiens. In this work, we evaluated the expression of differentially expressed genes (DEGs), proteins, and transcription factors (TFs) in three different types of cells in mice: antibody-secreting cells, cardiac mesenchymal stromal cells, and skeletal muscle cells. The goal of this article is to uncover a common cause during aging among these cells in order to increase understanding about establishing complete techniques for preventing aging and improving people's quality of life. We conducted a comprehensive network-based investigation to establish which genes and proteins are shared by the three different types of aged cells. Our findings clearly indicated that aging induces gene dysregulation in immune, pharmacological, and apoptotic pathways. Furthermore, our research developed a list of hub genes with differential expression in aging responses that should be investigated further to discover viable anti-aging treatments.

PHF14 enhances DNA methylation of SMAD7 gene to promote TGF-β-driven lung adenocarcinoma metastasis

Aberrant activation of TGF-β signaling plays a pivotal role in cancer metastasis and progression. However, molecular mechanisms underlying the dysregulation of TGF-β pathway remain to be understood. Here, we found that SMAD7, a direct downstream transcriptional target and also a key antagonist of TGF-β signaling, is transcriptionally suppressed in lung adenocarcinoma (LAD) due to DNA hypermethylation. We further identified that PHF14 binds DNMT3B and serves as a DNA CpG motif reader, recruiting DNMT3B to the SMAD7 gene locus, resulting in DNA methylation and transcriptional suppression of SMAD7. Our in vitro and in vivo experiments showed that PHF14 promotes metastasis through binding DNMT3B to suppress SMAD7 expression. Moreover, our data revealed that PHF14 expression correlates with lowered SMAD7 level and shorter survival of LAD patients, and importantly that SMAD7 methylation level of circulating tumor DNA (ctDNA) can potentially be used for prognosis prediction. Together, our present study illustrates a new epigenetic mechanism, mediated by PHF14 and DNMT3B, in the regulation of SMAD7 transcription and TGF-β-driven LAD metastasis, and suggests potential opportunities for LAD prognosis.

An autophagy-related diagnostic biomarker for uterine fibroids: FOS.

Uterine fibroids (UFs) are the most common benign gynecologic tumors in reproductive-aged women. The typical diagnostic strategies of UFs are transvaginal ultrasonography and pathological feature, while molecular biomarkers are considered conventional options in the assessment of the origin and development of UFs in recent years. Here, we extracted the differential expression genes (DEGs) and differential DNA methylation genes (DMGs) of UFs from the Gene Expression Omnibus (GEO) database, GSE64763, GSE120854, GSE45188, and GSE45187. 167 DEGs with aberrant DNA methylation were identified, and further Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed by the relevant R package. We next discerned 2 hub genes (FOS, and TNFSF10) with autophagy involvement by overlapping 167 DEGs and 232 autophagic regulators from Human Autophagy Database. FOS was identified as the most crucial gene through the Protein-Protein Interactions (PPI) network with the correlation of the immune scores. Moreover, the down-regulated expression of FOS in UFs tissue at both mRNA and protein levels was validated by RT-qPCR and immunohistochemistry respectively. The area under the ROC curve (AUC) of FOS was 0.856, with a sensitivity of 86.2% and a specificity of 73.9%. Overall, we explored the possible biomarker of UFs undergoing DNA-methylated autophagy and provided clinicians with a comprehensive assessment of UFs.

DNA methylation signatures of duplicate gene evolution in angiosperms.

Gene duplication is a source of evolutionary novelty. DNA methylation may play a role in the evolution of duplicate genes (paralogs) through its association with gene expression. While this relationship has been examined to varying extents in a few individual species, the generalizability of these results at either a broad phylogenetic scale with species of differing duplication histories or across a population remains unknown. We applied a comparative epigenomics approach to 43 angiosperm species across the phylogeny and a population of 928 Arabidopsis (Arabidopsis thaliana) accessions, examining the association of DNA methylation with paralog evolution. Genic DNA methylation was differentially associated with duplication type, the age of duplication, sequence evolution, and gene expression. Whole genome duplicates were typically enriched for CG-only gene-body methylated or unmethylated genes, while single-gene duplications were typically enriched for non-CG methylated or unmethylated genes. Non-CG methylation, in particular, was characteristic of more recent single-gene duplicates. Core angiosperm gene families differentiated into those which preferentially retain paralogs and 'duplication-resistant' families, which convergently reverted to singletons following duplication. Duplication-resistant families that still have paralogous copies were, uncharacteristically for core angiosperm genes, enriched for non-CG methylation. Non-CG methylated paralogs had higher rates of sequence evolution, higher frequency of presence-absence variation, and more limited expression. This suggests that silencing by non-CG methylation may be important to maintaining dosage following duplication and be a precursor to fractionation. Our results indicate that genic methylation marks differing evolutionary trajectories and fates between paralogous genes and have a role in maintaining dosage following duplication.

Abstract LB046: Characterizing the role of inflammation-induced epigenetic alterations in modulating the immune microenvironment during lung cancer initiation

Abstract One of the most important goals of developing timely detection and effective intervention strategies for lung cancer is to understand the biology and molecular mechanisms involved in early-stage evolution of this disease. Chronic inflammation is a major driver of lung cancer and contributes to its pathogenesis by inducing genetic and epigenetic abnormalities, as well as shifts in key immune cell populations. However, the evolution of these changes and the mechanisms underlying the early stages in development of pre-neoplasias and progression to invasive tumors have not been well delineated. Monolayer cell culture systems, while providing valuable insights into lung cancer development, lack the complexity of an intact organ system. This study aims to overcome these limitations by using a 3D lung organoid model to delineate the role of epigenetic alterations in driving initiation of non-small cell lung cancer. The stem cell enriched nature of the organoids makes it an ideal system to evaluate the implications of epigenetic alterations in these key cell types and their role in cancer initiation. To better identify the epigenetic alterations that play key roles in lung cancer initiation, cigarette smoke condensate (CSC) was applied to normal lung organoids to mimic chronic inflammatory exposure. Epigenomic and transcriptomic alterations were evaluated by genome-wide DNA methylation analysis, ATAC-seq, and RNA-seq. Flow cytometry and confocal microscopy were used to evaluate changes in composition of cell populations comprising the organoids. Long-term CSC exposure caused distinct morphological changes in organoid structure and cellular composition, accompanied by an increased proliferative potential. Analysis of changes in cell composition revealed increases in Krt14 expression in CSC treated organoids, leading to key shifts in basal stem cell populations from a TP63+KRT5+ to a TP63+KRT5+KRT14+ population. This was accompanied by reduction in differentiated cell types, as analyzed by qRT-PCR. Analysis of genome wide DNA methylation showed increases in promoter DNA methylation in key genes associated with lung tumorigenesis. Most importantly, co-culture studies involving culturing CSC treated and control organoids with key immune cells revealed that chronic CSC treatment caused modulation of the microenvironment from a pro- to an anti-inflammatory state. Our results suggest that chronic inflammation causes key shifts in populations of lung stem cells with an associated decrease in differentiation potential. These changes are accompanied by DNA methylation and gene expression changes suggestive of an increased tumorigenic potential. Finally, these changes in the CSC treated organoids are associated with the ability to modulate the function of key immune cells associated with lung tumorigenesis from a pro- to an anti-inflammatory phenotype. Results from our study, will aid in developing novel biomarker-based methodologies to distinguish and treat lung cancer in its early stages. Citation Format: Na Wang, Ray-Whay Chiu Yen, Hamza Khan, Malcolm V. Brock, Hariharan Easwaran, Stephen B. Baylin, Michelle Vaz. Characterizing the role of inflammation-induced epigenetic alterations in modulating the immune microenvironment during lung cancer initiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB046.

DNA Demethylation of Myogenic Genes May Contribute to Embryonic Leg Muscle Development Differences between Wuzong and Shitou Geese.

Skeletal muscle development from embryonic stages to hatching is critical for poultry muscle growth, during which DNA methylation plays a vital role. However, it is not yet clear how DNA methylation affects early embryonic muscle development between goose breeds of different body size. In this study, whole genome bisulfite sequencing (WGBS) was conducted on leg muscle tissue from Wuzong (WZE) and Shitou (STE) geese on embryonic day 15 (E15), E23, and post-hatch day 1. It was found that at E23, the embryonic leg muscle development of STE was more intense than that of WZE. A negative correlation was found between gene expression and DNA methylation around transcription start sites (TSSs), while a positive correlation was observed in the gene body near TTSs. It was also possible that earlier demethylation of myogenic genes around TSSs contributes to their earlier expression in WZE. Using pyrosequencing to analyze DNA methylation patterns of promoter regions, we also found that earlier demethylation of the MyoD1 promoter in WZE contributed to its earlier expression. This study reveals that DNA demethylation of myogenic genes may contribute to embryonic leg muscle development differences between Wuzong and Shitou geese.

In silico analysis of differentially expressed-aberrantly methylated genes in breast cancer for prognostic and therapeutic targets.

Breast cancer (BC) is the leading cause of death among women across the globe. Abnormal gene expression plays a crucial role in tumour progression, carcinogenesis and metastasis of BC. The alteration of gene expression may be through aberrant gene methylation. In the present study, differentially expressed genes which may be regulated by DNA methylation and their pathways associated with BC have been identified. Expression microarray datasets GSE10780, GSE10797, GSE21422, GSE42568, GSE61304, GSE61724 and one DNA methylation profile dataset GSE20713 were downloaded from Gene Expression Omnibus database (GEO). Differentially expressed-aberrantly methylated genes were identified using online Venn diagram tool. Based on fold change expression of differentially expressed-aberrantly methylated genes were chosen through heat map. Protein-protein interaction (PPI) network of the hub genes was constructed by Search Tool for the Retrieval of Interacting Genes (STRING). Gene expression and DNA methylation level of the hub genes were validated through UALCAN. Overall survival analysis of the hub genes was analysed through Kaplan-Meier plotter database for BC. A total of 72 upregulated-hypomethylated genes and 92 downregulated-hypermethylated genes were obtained from GSE10780, GSE10797, GSE21422, GSE42568, GSE61304, GSE61724, and GSE20713 datasets by GEO2R and Venn diagram tool. PPI network of the upregulated-hypomethylated hub genes (MRGBP, MANF, ARF3, HIST1H3D, GSK3B, HJURP, GPSM2, MATN3, KDELR2, CEP55, GSPT1, COL11A1, and COL1A1) and downregulated-hypermethylated hub genes were constructed (APOD, DMD, RBPMS, NR3C2, HOXA9, AMKY2, KCTD9, and EDN1). All the differentially expressed hub genes expression was validated in UALCAN database. 4 in 13 upregulated-hypomethylated and 5 in 8 downregulated-hypermethylated hub genes to be significantly hypomethylated or hypermethylated in BC were confirmed using UALCAN database (p < 0.05). MANF, HIST1H3D, HJURP, GSK3B, GPSM2, MATN3, KDELR2, CEP55, COL1A1, APOD, RBPMS, NR3C2, HOXA9, ANKMY2, and EDN1 were significantly (p < 0.05) associated with poor overall survival (OS). The identified aberrantly methylated-differentially expressed genes and their related pathways and function in BC can serve as novel diagnostic and prognostic biomarkers and therapeutic targets.Please confirm if the author names are presented accurately and in the correct sequence (given name, middle name/initial, family name). Author 4 Given name: [Jeewan Ram] Last name [Vishnoi]. Also, kindly confirm the details in the metadata are correct.It is correct.

The Effect of Metformin and Carbohydrate-Controlled Diet on DNA Methylation and Gene Expression in the Endometrium of Women with Polycystic Ovary Syndrome

Polycystic ovary syndrome (PCOS) is an endocrine disease associated with infertility and metabolic disorders in reproductive-aged women. In this study, we evaluated the expression of eight genes related to endometrial function and their DNA methylation levels in the endometrium of PCOS patients and women without the disease (control group). In addition, eight of the PCOS patients underwent intervention with metformin (1500 mg/day) and a carbohydrate-controlled diet (type and quantity) for three months. Clinical and metabolic parameters were determined, and RT-qPCR and MeDIP-qPCR were used to evaluate gene expression and DNA methylation levels, respectively. Decreased expression levels of HOXA10, GAB1, and SLC2A4 genes and increased DNA methylation levels of the HOXA10 promoter were found in the endometrium of PCOS patients compared to controls. After metformin and nutritional intervention, some metabolic and clinical variables improved in PCOS patients. This intervention was associated with increased expression of HOXA10, ESR1, GAB1, and SLC2A4 genes and reduced DNA methylation levels of the HOXA10 promoter in the endometrium of PCOS women. Our preliminary findings suggest that metformin and a carbohydrate-controlled diet improve endometrial function in PCOS patients, partly by modulating DNA methylation of the HOXA10 gene promoter and the expression of genes implicated in endometrial receptivity and insulin signaling.

Integrative Analysis of DNA Methylation and Gene Expression Data Identifies Potential Biomarkers and Functional Epigenetic Modules for SARS-CoV-2.

To integrate gene expression and DNA methylation data and find the potential role of DNA methylation in the invasion and replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We first conducted differential expression and methylation analysis between the coronavirus disease of 2019 (COVID-19) and healthy controls. FEM was employed to identify functional epigenetic modules, from which a diagnostic model for COVID-19 was built. SKA1 and WSB1 modules were identified, with SKA1 module enriched in COVID-19 replication and transcription, and WSB1 module related to ubiquitin-protein activity. The differentially expressed or differentially methylated genes in these two modules could be used to distinguish COVID-19 from healthy controls, with AUC reaching 1 and 0.98 for SKA1 and WSB1 modules, respectively. Two epigenetically activated genes (CENPM and KNL1) from the SKA1 module were upregulated in HPV- or HBV-positive tumor samples and were found to be significantly associated with the survival of tumor patients. In conclusion, the identified FEM modules and potential signatures play an essential role in the replication and transcription of coronavirus.

Abstract 6014: Cancer risk diagnosis by epimutation burden: a multicenter prospective study

Abstract Aberrant DNA methylation, induced by aging and accelerated by chronic inflammation, accumulates in normal tissue. Accumulation of DNA methylation in multiple genes, namely epimutation burden, is closely associated with cancer risk. Indeed, we demonstrated that the measurement of the methylation level of a marker gene in normal tissue is useful for cancer risk prediction of metachronous cancers, by a multicenter prospective study with gastric cancer patients (Asada, Gut, 64:388, 2015: Maeda, Gut, 66:1721, 2017). In this study, we attempted to predict the risk of primary gastric cancer by performing a new multicenter prospective study. We recruited 1,880 healthy people, after Helicobacter pylori eradication, with a clinically high risk of gastric cancer. Biopsy specimens were obtained from the antrum and body, and the methylation level of RIMS1, which is a preselected epigenomic risk marker for this study, was measured in 1,757 participants. The participants are annually being followed-up to detect primary gastric cancer, for 5-8 years. Currently, the median follow-up is 4.05 years, and 27 people have developed primary gastric cancers (as of September 2022). The results of statistical analysis are being determined. This study will establish the clinical utility of a cancer risk diagnosis by epimutation burden in normal tissue, and is promising for application in various inflammation-associated cancers. Citation Format: Harumi Yamada, Masahiro Maeda, Seiichiro Abe, Taichi Shimazu, Takayuki Ando, Kazunari Murakami, Shinji Tanaka, Takao Maekita, Osamu Inatomi, Ken Sugimoto, Tetsuro Kawagoe, Nobutake Yamamichi, Toshikazu Ushijima. Cancer risk diagnosis by epimutation burden: a multicenter prospective study. [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 6014.

Abstract 202: Perfused patient-derived tumor organoid models with autologous immune cells for preclinical drug development

Abstract The paradigm shift towards inclusion of primary patient samples in preclinical drug development bottlenecks at the tissue procurement step due to an on-going lack of ’research-ready’ tumor samples. Organoid technology was developed as a solution to this hurdle. Their ability to expand in culture makes them a renewable resource similar to cell lines while recapitulating key properties of the primary tissue, including tumor heterogeneity, structural organization and drug responses. As preclinical drug development continues to adopt organoid-based screening technology and immune-oncology agents continue to be advanced, there is high demand for more complex organoid-based models capable of more accurately predicting individual patient drug responses. Due to their development process, the vast majority of organoids do not contain immune cells, resulting in the need to utilize allogeneic immune cells for any immune cell involved interrogation of the models. Maintaining the immune cells for incorporation with the organoid model can overcome this hindrance. Additionally, perfused organoid models offer a complex, ex vivo model with the ability to replicate the mechanical forces imparted by blood flow and interstitial pressure. Compared to 2D and static 3D cultures, perfused 3D culture models show enhanced proliferative and migratory capacities and can be sustained as long-term cultures. In our previous studies, DNA methylation signatures in patient-derived breast microtumors grown in Kiyatec’s 3DKUBE™ perfusion bioreactor matched the primary tissues of derivation. Given the need for more complex biology when assessing immune-oncology agents, the physiological relevance in maintaining gene expression ex vivo, and the capability to sustain long-term viability, the impact of autologous immune cells and perfusion on the success of organotypic culture was investigated across a panel of primary patient tumors. Immune cells were profiled for status and activity and imaging and viability assays were used to evaluate static and perfused 3D organoid models for differences in viability and morphology. PCR was used to examine how mechanically transduced forces alter gene expression and DNA methylation signatures. Drug responses were measured using our KIYA-Predict™ platform, comparing organoid drug responses to primary tissue drug responses. Results of this study provide insight on alternative methods of organotypic culture and evidence for the utilization of more complex tissue modeling in preclinical drug development. Citation Format: Melissa Millard, Natalie Dance, Teresa M. DesRochers. Perfused patient-derived tumor organoid models with autologous immune cells for preclinical drug development [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 202.

Abstract 4266: Genomic landscape of prostate cancer: Bioinformatic analysis of TCGA PanCancer data by race and identification of key pathways and genes in PTEN mutation

Abstract Issue: Prostate cancer (Pca) is still among the most frequently diagnosed cancers in adults. The most often mutated gene in PCa is PTEN (Phosphatase and Tensin Homolog), making it a possible biomarker for prospective customized therapy. The root causes of health inequalities in PCa have not received much attention. Numerous online databases, like The Cancer Genome Atlas (TCGA), provide gene expression data and other data, allowing researchers to further investigate new processes or therapy options. However, there hasn't been much study on tailored therapy up to this point since there may have been difficulties with access to genomic information and bioinformatic tools. Methods: The PCa data in cBioPortal comprises 494 samples. We selected 154 samples comprising 7 Blacks and 147 Whites. We used the TCGA database to obtain the PCa gene expression and DNA methylation data for study. In order to examine the data by race/ethnicity, gene expression, DNA methylation, and genomic changes, we evaluated at the differentially expressed and methylated genes using online tools (cBioPortal). To evaluate gene methylation, expression, and alterations, log ratios of means in Blacks to means in Whites were computed across the data. Additionally, Kaplan-Meier survival analysis, protein interaction analysis, and multivariable data analysis were carried out. Results: PCa data in the TCGA database showed PTEN mutation in 20% patients. The median age of participants was 63 years (range: 42-78). For every category of data assessed, there were significant differences by race/ethnicity (q &amp;lt; 0.05). In the survival analysis, SLC2A1, NUDT12, CUTALP, and other genes were associated with survival. Conclusion: Genetic alteration, expression, and DNA methylation are each significantly different by the two race groups compared. According to our research, PTEN mutation in PCa may influence several genes and biological processes. Citation Format: Olumide Arigbede, Sarah G. Buxbaum, Sara Falzarano, Suhn K. Rhie. Genomic landscape of prostate cancer: Bioinformatic analysis of TCGA PanCancer data by race and identification of key pathways and genes in PTEN mutation. [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 4266.

Abstract 1005: LINE-1 hypomethylation is associated with poor outcomes in locoregionally advanced oropharyngeal cancer

Abstract Background: Currently, human papillomavirus (HPV) positivity represents a strong prognostic factor for both reduced risk of relapse and improved survival in patients with oropharyngeal squamous cell carcinoma (OPSCC). However, a subset of HPV-positive OPSCC patients still experience poor outcomes. Furthermore, HPV-negative OPSCC patients, who have an even higher risk of relapse, are still lacking suitable prognostic biomarkers for clinical outcome. Here, we evaluated the prognostic value of LINE-1 methylation level in OPSCC patients, and further addressed the relationship between LINE-1 methylation status and p53 protein expression as well as genome-wide/gene-specific DNA methylation. Results: In this study, DNA was extracted from 163 formalin-fixed paraffin-embedded tissue samples retrospectively collected from stage III-IVB OPSCC patients managed with curative intent with up-front treatment. Quantitative methylation-specific PCR revealed that LINE-1 hypomethylation was directly associated with poor prognosis (5-year overall survival - OS: 28.1% for LINE-1 methylation&amp;lt;35% versus 69.1% for ≥55%; p&amp;lt;0.0001). When LINE-1 methylation was dichotomized as &amp;lt;55% vs. ≥55%, interaction with HPV16 emerged: compared with hypermethylated HPV-16 positive patients, subjects with hypomethylated HPV16-negative OPSCC reported an adjusted higher risk of death (HR: 4.83, 95% CI: 2.24-10.38) and progression (HR: 4.54, 95% CI: 2.18-9.48). Tumor protein p53 (TP53) gene is often mutated and overexpressed in HPV-negative OPSCC. Since p53 has been reported to repress LINE-1 promoter, we then analyzed the association between p53 protein expression and LINE-1 methylation levels. Following p53 immunohistochemistry, results indicated that among HPV16-negative patients with p53≥50%, LINE-1 methylation levels declined and remained stable at approximately 43%; any HPV16-positive patient reported p53≥50%. Finally, DNA methylation analysis demonstrated that genome-wide average methylation level at cytosine-phosphate-guanine sites was significantly lower in HPV16-negative OPSCC patients who relapsed within two years. The subsequent integrative analysis of gene expression and DNA methylation identified 20 up-regulated/hypomethylated genes in relapsed patients, and most of them contained LINE-1 elements in their promoter sequences. Conclusions: Evaluation of the methylation level of LINE-1 may help in identifying the subset of OPSCC patients with bad prognosis regardless of their HPV status. Aberrant LINE-1 hypomethylation might occur along with TP53 mutations, and lead to altered gene expression in OPSCC. Citation Format: Mariateresa Casarotto, Valentina Lupato, Giorgio Giurato, Roberto Guerrieri, Sandro Sulfaro, Annamaria Salvati, Elisa D'Angelo, Carlo Furlan, Anna Menegaldo, Lorena Baboci, Barbara Montico, Irene Turturici, Riccardo Dolcetti, Salvatore Romeo, Vittorio Baggio, Stefania Corrado, Gianluca Businello, Maria Guido, Alessandro Weisz, Vittorio Giacomarra, Giovanni Franchin, Agostino Steffan, Luca Sigalotti, Emanuela Vaccher, Paolo Boscolo-Rizzo, Jerry Polesel, Giuseppe Fanetti, Elisabetta Fratta. LINE-1 hypomethylation is associated with poor outcomes in locoregionally advanced oropharyngeal 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 1005.

Abstract 5999: Comparative and integrative analysis of transcriptomic and epigenome wide DNA methylation changes in African American prostate cancer

Abstract African American (AA) men have the highest incidence and mortality rate from Prostate cancer (PCa) than any other racial/ethnic group. To understand this racial disparity, molecular features of different types, including gene expression, DNA methylation and other genomic alterations, must be carried out in tumor samples from these 2 populations. To date, PCa genomic studies have largely under-represented tumor samples from AA men due to lack of inclusion of significant numbers in such studies. In this study we carried out genome-wide DNA methylation analysis in 63 clinically annotated fresh-frozen PCa and 50 normal prostate tissues from AA men using the Illumina Infunium 850K Human Methylation EPIC BeadChip array. mRNA expression database from a sub-set of the AA biospecimen were used to assess correlation of transcriptome and epigenomic datasets. Using bioinformatic analysis to integrate a comprehensive set of genomic datasets we identified probesets that were significant (p&amp;lt;0.01) and differentially methylated in AA PCa compared to normal prostate tissues and were significant (p&amp;lt;0.01) and inversely correlated with AA mRNA expression. GO analysis in this AA cohort showed significant differential methylation that were mostly hypermethylated genes with corresponding down-regulated mRNA expression for top genes associated with biological pathways in hemidesmosome assembly, mammary gland development, epidermis development, hormone biosynthetic process and cell communication. On the other hand, few significant differential top hypomethylated genes and corresponding up-regulated mRNA expression were identified in biological pathways of negative regulation of macrophage differentiation, cAMP-dependent protein kinase inhibitor activity, protein destabilization, transcription corepressor activity and fatty acid biosynthetic process. Furthermore, we identified significant and differential methylated genes in our AA PCa cohort that were associated with PCa progression when compared to the TCGA EA and AA dataset. These genes were AMIGO3, IER3, UPB1, GRM7, TFAP2C, TOX2, PLSCR2, ZNF292, ESR2, MIXL1, BOLL and FGF6. Overall, IPA of differential methylated promoter region are genes enriched for network involved in sex-steroid hormonal signals and tumor microenvironment signaling pathways. Our integrative analysis provides new candidate genes associated with prostate cancer progression in AA men as potential targets for improving prostate cancer treatment and addressing the racial disparity problem. Citation Format: Bernard Kwabi-Addo, Somiranjan Ghosh, Chad Creighton, Michael Ittmann. Comparative and integrative analysis of transcriptomic and epigenome wide DNA methylation changes in African American prostate 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 5999.

Abstract 5352: Targeting DNA methylation in T cells to improve the efficacy of immunotherapy

Abstract T-cells are critical mediators of immunity and immunologic memory. Their cell fates are regulated in part through epigenetic mechanisms, including DNA methylation. Recent genome-wide methylation analyses have revealed dynamic alterations in the methylome at various stages of development and differentiation of T cells. At single cell level, it is not easy to simultaneously collect RNA-seq and RBBS methylation profiling. An important task is to understand the expression change of which genes and pathways are regulated by DNA methylations, especially for the ones that are associated with functional variations in the T cells from tumor microenvironment.In this study, we developed a computational approach based on our recently developed metabolic flux estimation to estimate cell-wise global DNA methylation activity level by using scRNA-seq data. We also hypothesize that the global DNA methylation activity level in one cell determines most of the DNA methylation level in gene-specific DNA methylations. Hence, the dependency between gene-specific DNA methylation and expression could be imputed by the dependency between predicted global DNA methylation input level and the gene expression. We validated our method to impute cell-wise global DNA methylation level by using four independent sets of paired gene expression and DNA methylation data. Noted, our prediction of global DNA methylation activity is from a pure metabolic perspective. We found that two metabolic reaction rates, named metabolic flux from methionine to SAM and SAM to SAH, purely predicted by using gene expression data can accurately impute DNA methylation activity in all validating data sets. Our method enables further identification of the disease/cell context specific contributor of DNA methylation, i.e., the genes high contribution to DNA methylations in each individual cell or cell groups. We applied our method on scRNA-seq data of different T cell types extracted from TME of lung, liver, and colon cancer. We have seen that exhausted T cells, especially the ones with decreased Granzymes and PRF1 are associated with increased global DNA methylation level and related genes, suggesting the potential clinical implications in targeting DNA methylation to improve the efficacy of immunotherapies. Citation Format: Pengtao Dang, Xiao Wang, Haiqi Zhu, Jia Wang, Tingbo Guo, Xinyu Zhou, Paveethran Swaminathan, Chi Zhang, Sha Cao. Targeting DNA methylation in T cells to improve the efficacy of immunotherapy. [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 5352.

Abstract 4307: Integrative molecular subtypes of acute myeloid leukemia

Abstract Introduction: Acute Myeloid Leukemia (AML) is a heterogenous disease characterized by distinct clinical course and prognosis based on genomics, epigenomics or transcriptomics profile. However, the multi-omics profiles have not been systematically integrated to define the integrative molecular subtypes of AML. There is a great clinical interest to identify the patterns across multi-omics profiles that could be used for prognosis and guiding targeted therapy in AML. Methods: To identify integrative molecular subtypes (iSubtypes) of AML, we performed an integrative clustering (iCluster) analysis of the AML multi-omics data (n=160) generated by The Cancer Genome Atlas (TCGA), which consisted of somatic mutation, DNA copy number, DNA methylation and RNA-seq gene expression data for 160 AML samples. We identified the multi-omics signatures that drove the molecular classification of AML. Based on the methylation and gene expression signatures, we derived a gene panel for classification of AML and verified the prognostic power of the gene panel using three independent AML datasets (n=1382). Results: We identified four iSubtypes of AML that featured distinct multi-omics signatures. At the DNA level, the iSubtype 1 was characterized by chromosomal abnormalities and high-frequency mutation of TP53 (30%) and RUNX1 (27%); the iSubtype 2 was characterized by chromosomal abnormalities and high-frequency mutation of CEBPA (20%) and FLT3 (26%); while the iSubtypes 3 and 4 were characterized by chromosomal normality and high-frequency mutation of FLT3 (34%, 41%), NPM1 (37%, 57%) and DNMT3A (20%, 41%). At the epigenomics level, the iSubtypes 1, 3 and 4 were characterized by hypomethylation of subtype driver genes, while the iSubtypes 2 was characterized by hypermethylation of subtype driver genes. At the transcriptomics level, the 4 iSubtypes were distinguished by differentially expressed genes involved in immune process, regulation of immune process, angiogenesis, leukocyte cell activation and migration, wound healing and cell structure organization, etc. The iSubtypes 1 and 4 had the worse overall survival (OS), while the iSubtype 3 had the best and the iSubtype 2 had the middle OS. Based on the subtype-driving methylation and transcription signatures, we derived a 571-gene panel for classification of AML. Using 3 independent AML transcriptomics datasets, we demonstrated that the excellent power of the gene panel in classifying AML into 4 gene expression subtypes with similar survival outcomes. Conclusions: iSubtypes of AML are jointly determined by multi-omics profiles. The subtype-driving methylation and transcription signature have excellent power in classifying AML into clinically relevant subtypes. This integrative analysis provides insights into the biological processes underling AML. Citation Format: Qianxing (Quincy) Mo, Seongseok Yun, David A. Sallman, Nicole Vincelette, Guang Peng, Ling Zhang, Jeffrey E. Lancet, Eric Padron. Integrative molecular subtypes of acute myeloid leukemia. [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 4307.

Molecular Subtyping and Survival Analysis of Osteosarcoma Reveals Prognostic Biomarkers and Key Canonical Pathways.

Osteosarcoma (OS) is a common bone malignancy in children and adolescents. Although histological subtyping followed by improved OS treatment regimens have helped achieve favorable outcomes, a lack of understanding of the molecular subtypes remains a challenge to characterize its genetic heterogeneity and subsequently to identify diagnostic and prognostic biomarkers for developing effective treatments. In the present study, global analysis of DNA methylation, and mRNA and miRNA gene expression in OS patient samples were correlated with their clinical characteristics. The mucin family of genes, MUC6, MUC12, and MUC4, were found to be highly mutated in the OS patients. Results revealed the enrichment of molecular pathways including Wnt signaling, Calcium signaling, and PI3K-Akt signaling in the OS tumors. Survival analyses showed that the expression levels of several genes such as RAMP1, CRIP1, CORT, CHST13, and DDX60L, miRNAs and lncRNAs were associated with survival of OS patients. Molecular subtyping using Cluster-Of-Clusters Analysis (COCA) for mRNA, lncRNA, and miRNA expression; DNA methylation; and mutation data from the TARGET dataset revealed two distinct molecular subtypes, each with a distinctive gene expression profile. Between the two subtypes, three upregulated genes, POP4, HEY1, CERKL, and seven downregulated genes, CEACAM1, ABLIM1, LTBP2, ISLR, LRRC32, PTPRF, and GPX3, associated with OS metastasis were found to be differentially regulated. Thus, the molecular subtyping results provide a strong basis for classification of OS patients that could be used to develop better prognostic treatment strategies.