CpG Island Methylation Research Articles

Differential expression of ADRB1 causes different responses to norepinephrine in adipocytes of Duroc-Landrace-Yorkshire pigs and min pigs

Research has shown that pigs from different regions exhibit varying responses to cold stimuli. Typically, cold stimuli induce browning of white adipose tissue mediated by adrenaline, promoting non-shivering thermogenesis. However, the molecular mechanisms underlying differential response of pig breeds to norepinephrine are unclear.The aim of this study was to investigate the differences and molecular mechanisms of the effects of norepinephrine (NE) treatment on adipocytes of Min pigs (a cold-resistant pig breed) and Duroc-Landrace-Yorkshire (DLY) pigs. Real time-qPCR, western blot, and immunofluorescence were performed following NE treatment on cell cultures of adipocytes originating from Min pigs (n = 3) and DLY pigs (n = 3) to assess the expressions of adipogenesis markers, beige fat markers, and mitochondrial biogenesis markers. The results showed that NE did not affect browning of adipocytes in DLY pigs, whereas promoted browning of adipocytes in Min pigs. Further, the expression of ADRB1 (Adrenoceptor Beta 1, ADRB1) was higher in subcutaneous adipose tissue and adipocytes of Min pigs than those of DLY pigs. Overexpression of ADRB1 in DLY pig adipocytes enhanced sensitivity to NE, exhibiting decreased adipogenesis markers, upregulated beige fat markers, and increased mitochondrial biogenesis. Conversely, adipocytes treated with ADRB1 antagonist in Min pigs resulted in decreased cellular sensitivity to NE. Further studies revealed differential CpG island methylation in ADRB1 promoter region, with lower methylation levels in Min pigs compared to DLY pigs. In conclusion, differential methylation of the ADRB1 promoter region leads to different ADRB1 expression, resulting in varying responsiveness to NE in adipocytes of two pig breeds. Our results provide new insights for further analysis of the differential cold responsiveness in pig breeds from different regions.

Pathological remodeling of reactive astrocytes: Involvement of DNA methylation and downregulation of homeostatic genes.

Astrocytes provide metabolic support to neurons, maintain ionic and water homeostasis, and uptake and recycle neurotransmitters. After exposure to the prototypical PAMP lipopolysaccharide (LPS), reactive astrocytes increase the expression of pro-inflammatory genes, facilitating neurodegeneration. In this study, we analyzed the expression of homeostatic genes in astrocytes exposed to LPS and identified the epigenetic factors contributing to the suppression of homeostatic genes in reactive astrocytes. Primary astrocytic cultures were acutely exposed to LPS and allowed to recover for 24, 72 h, and 7 days. As expected, LPS exposure induced reactive astrogliosis and increased the expression of pro-inflammatory IL-1B and IL-6. Interestingly, the acute exposure resulted in persistent hypermethylation of astroglial DNA. Similar hypermethylation was observed in highly reactive astrocytes from the traumatic brain injury (TBI) penumbra invivo. Hypermethylation was accompanied by decreased expression of homeostatic genes including LDHA and Scl16a1 (MCT1) both involved in the lactate shuttle to neurons; glutamine synthase (GS) responsible for glutamate processing; Kcnj10 (Kir4.1) important for K+ homeostasis, and the water channel aquaporin-4 (Aqp4). Furthermore, the master regulator of DNA methylation, MAFG-1, as well as DNA methyl transferases DNMT1 and DNMT3a were overexpressed. The downregulation of homeostatic genes correlated with increased methylation of CpG islands in their promoters, as assessed by methylation-sensitive PCR and increased DNMT3a binding to the GS promoter. Treatment with decitabine, a DNMT inhibitor, prevented the LPS- and the HMGB-1-induced downregulation of homeostatic genes. Decitabine treatment also prevented the neurotoxic effects of these astrocytes in primary cortical cultures. In summary, our findings reveal that the pathological remodeling of reactive astrocytes encompasses not only the pro-inflammatory response but, significantly, also entails a long-term suppression of homeostatic gene expression with methylation of crucial CpG islands within their promoters.

HGG-04. MALIGNANT GLIOMA IN L-2-HYDROXY GLUTARIC ACIDURIA; THOROUGH MOLECULAR CHARACTERIZATION OF A CASE AND LITERATURE REVIEW

Abstract L-2-hydroxyglutaric aciduria (L-2-HGA) is a rare neurometabolic disorder characterized by accumulation of L2-hydroxyglutarate (L-2-HG) due to mutations in the L2HGDH gene. L-2-HGA patients have a significantly increased lifetime risk of central nervous system (CNS) tumors. Here, we present a 16-year-old girl with L-2-HGA who developed a tumor in the right cerebral hemisphere, which was discovered after left-sided neurological deficits of the patient. Histologically, the tumor had a high-grade diffuse glioma phenotype. DNA sequencing revealed the germline L2HGDH mutation as well as inactivating mutations in TP53, BCOR and NF1. Genome-wide DNA-methylation analysis was unable to classify the tumor with high confidence. More detailed analysis revealed that this tumor clustered amongst IDH-wildtype gliomas by methylation profiling and did not show the glioma CpG island methylator phenotype (G-CIMP) (in contrast to IDH-mutant diffuse gliomas with accumulated levels of D-2-HG). These findings were against all our expectations given the inhibitory potential of 2-HG on DNA-demethylation enzymes. Our final integrated histomolecular diagnosis was diffuse pediatric-type high-grade glioma, H3-wildtype and IDH-wildtype. Due to rapid tumor progression the patient died nine months after initial diagnosis. In this manuscript we provide extensive molecular characterization of the tumor as well as a literature review focusing on oncogenetic considerations of L-2-HGA-associated CNS tumors.

Unmasking early colorectal cancer clues: in silico and in vitro investigation of downregulated IGF2, SOCS1, MLH1, and CACNA1G in SSA polyps

Background and aimColorectal cancer (CRC) originates from pre-existing polyps in the colon. The development of different subtypes of CRC is influenced by various genetic and epigenetic characteristics. CpG island methylator phenotype (CIMP) is found in about 15–20% of sporadic CRCs and is associated with hypermethylation of certain gene promoters. This study aims to find prognostic genes and compare their expression and methylation status as potential biomarkers in patients with serrated sessile adenomas/polyps (SSAP) and CRC, in order to evaluate which, one is a better predictor of disease.MethodThis study employed a multi-phase approach to investigate genes associated with CRC and SSAP. Initially, two gene expression datasets were analyzed using R and Limma package to identify differentially expressed genes (DEGs). Venn diagram analysis further refined the selection, revealing four genes from the Weissenberg panel with significant changes. These genes, underwent thorough in silico evaluations. Once confirmed, they proceeded to wet lab experimentation, focusing on expression and methylation status. This comprehensive methodology ensured a robust examination of the genes involved in CRC and SSAP.ResultThis study identified cancer-specific genes, with 8,351 and 1,769 genes specifically down-regulated in SSAP and CRC tissues, respectively. The down-regulated genes were associated with cell adhesion, negative regulation of cell proliferation, and drug response. Four highly downregulated genes in the Weissenberg panel, including CACNA1G, IGF2, MLH1, and SOCS1. In vitro analysis showed that they are hypermethylated in both SSAP and CRC samples while their expressions decreased only in CRC samples.ConclusionThis suggests that the decrease in gene expression could help determine whether a polyp will become cancerous. Using both methylation status and gene expression status of genes in the Weissenberg panel in prognostic tests may lead to better prognoses for patients.

Association between gut microbiota and CpG island methylator phenotype in colorectal cancer

ABSTRACT The intestinal microbiota is an important environmental factor implicated in CRC development. Intriguingly, modulation of DNA methylation by gut microbiota has been reported in preclinical models, although the relationship between tumor-infiltrating bacteria and CIMP status is currently unexplored. In this study, we investigated tumor-associated bacteria in 203 CRC tumor cases and validated the findings using The Cancer Genome Atlas datasets. We assessed the abundance of Bacteroides fragilis, Escherichia coli, Fusobacterium nucleatum, and Klebsiella pneumoniae through qPCR analysis and observed enrichment of all four bacterial species in CRC samples. Notably, except for E. coli, all exhibited significant enrichment in cases of CIMP. This enrichment was primarily driven by a subset of cases distinguished by high levels of these bacteria, which we labeled as “Superhigh”. The bacterial Superhigh status showed a significant association with CIMP (odds ratio 3.1, p-value = 0.013) and with MLH1 methylation (odds ratio 4.2, p-value = 0.0025). In TCGA CRC cases (393 tumor and 45 adj. normal), bacterial taxa information was extracted from non-human whole exome sequencing reads, and the bacterial Superhigh status was similarly associated with CIMP (odds ratio 2.9, p < 0.001) and MLH1 methylation (odds ratio 3.5, p < 0.001). Finally, 16S ribosomal RNA gene sequencing revealed high enrichment of Bergeyella spp. C. concisus, and F. canifelinum in CIMP-Positive tumor cases. Our findings highlight that specific bacterial taxa may influence DNA methylation, particularly in CpG islands, and contribute to the development and progression of CIMP in colorectal cancer.

Abstract B029: Assessing gene expression and methylation of KMT2D and IGF2 genes in patients with non-small cell lung cancer

Abstract Background: Aberrant promoter methylation of CpG islands is an important mechanism for regulation of gene expression. Recent data suggest that epigenetic abnormalities may occur very early in lung carcinogenesis. Systemic methylation changes may be a diagnostic marker for tumor development or prognosis. In this study, the expression and methylation of KMT2D and IGF2 genes were investigated in the lung cancer tissue compared to the adjacent normal tissue. Methods: The status of methylation of KMT2D and IGF2 genes were investigated in 30 patients with NSCLC after genomic DNA extraction using bisulfite treatment and MS-HRM method and the expression of these genes were checked by Real-Time PCR method in same samples. Results: For KMT2D gene, the expression and methylation level increased in 46.6% and 6.67% (respectively) for tumor samples comparison with normal samples (P&amp;gt;0.05). Also, for IGF2 gene 50% tumor samples overexpressed and 50% tumor samples showed that reduced expression comparison with the normal samples (P&amp;gt;0.05). In addition, 96.66% of tumor tissues did not show any change in methylation level for IGF2 gene promoter (P&amp;gt;0.05). Conclusion: This study showed that expression and methylation level of KMT2D and IGF2 genes did not change in NSCLC tumor samples compared to normal samples. However, this study was designed as a pilot study, and further investigations are required to confirm our findings. Citation Format: Arash Matinahmadi, Zoofa Zayani, Seyed Hesamoddin Bidooki, Alireza Tavakolpournegari. Assessing gene expression and methylation of KMT2D and IGF2 genes in patients with non-small cell lung cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr B029.

THY1 is a prognostic-related biomarker via mediating immune infiltration in lung squamous cell carcinoma (LUSC).

Thymus cell antigen 1 (THY1) has been proven to play pivotal roles in many diseases. However, we do not fully understand its functional mechanism, especially in lung squamous cell carcinoma (LUSC). Here, we aimed to perform a comprehensive analysis to explore the expression and prognostic values of THY1 in LUSC using bioinformatic technology. Some online public databases (e.g., ONCOMINE, PrognoScan, TIMER, Kaplan-Meier plotter, STRING, LinkedOmics, and GEPIA) were used to explore the expression, prognostic significance, and potential molecular mechanism of THY1. The analysis indicated that THY1 was significantly up-regulated and closely correlated with poor prognosis in many malignant tumors, including LUSC. Further analysis revealed that over-expression of THY1 was significantly correlated with clinicopathological parameters (e.g., individual cancer stage, age, smoking habits, nodal metastasis status, and TP53 mutation status) in LUSC. The CpG islands methylation of THY1 was negatively correlated with THY1 mRNA expression in The Cancer Genome Atlas Program (TCGA). Further enrichment analysis of THY1 correlated genes revealed that they were mainly correlated with the formation of extracellular matrix (ECM), and got involved in the pathway of epithelial mesenchymal transition (EMT). Furthermore, differentially expressed THY1 was significantly correlated with immune cell infiltrations and poor prognosis in LUSC. In summary, bioinformatic analysis demonstrated that THY1 was significantly over-expressed and closely correlated with unfavorable prognosis in LUSC, which may apply as a promising diagnostic and therapeutic biomarker for LUSC in the future.

Semimethylation is a feature of diffuse large B-cell lymphoma, and subgroups with poor prognosis are characterized by global hypomethylation and short telomere length

BackgroundLarge B-cell lymphoma (LBCL) is the most common lymphoma and is known to be a biologically heterogeneous disease regarding genetic, phenotypic, and clinical features. Although the prognosis is good, one-third has a primary refractory or relapsing disease which underscores the importance of developing predictive biological markers capable of identifying high- and low-risk patients. DNA methylation (DNAm) and telomere maintenance alterations are hallmarks of cancer and aging. Both these alterations may contribute to the heterogeneity of the disease, and potentially influence the prognosis of LBCL.ResultsWe studied the DNAm profiles (Infinium MethylationEPIC BeadChip) and relative telomere lengths (RTL) with qPCR of 93 LBCL cases: Diffuse large B-cell lymphoma not otherwise specified (DLBCL, n = 66), High-grade B-cell lymphoma (n = 7), Primary CNS lymphoma (n = 8), and transformation of indolent B-cell lymphoma (n = 12). There was a substantial methylation heterogeneity in DLBCL and other LBCL entities compared to normal cells and other B-cell neoplasms. LBCL cases had a particularly aberrant semimethylated pattern (0.15 ≤ β ≤ 0.8) with large intertumor variation and overall low hypermethylation (β > 0.8). DNAm patterns could not be used to distinguish between germinal center B-cell-like (GC) and non-GC DLBCL cases. In cases treated with R-CHOP-like regimens, a high percentage of global hypomethylation (β < 0.15) was in multivariable analysis associated with worse disease-specific survival (DSS) (HR 6.920, 95% CI 1.499–31.943) and progression-free survival (PFS) (HR 4.923, 95% CI 1.286–18.849) in DLBCL and with worse DSS (HR 5.147, 95% CI 1.239–21.388) in LBCL. These cases with a high percentage of global hypomethylation also had a higher degree of CpG island methylation, including islands in promoter-associated regions, than the cases with less hypomethylation. Additionally, telomere length was heterogenous in LBCL, with a subset of the DLBCL-GC cases accounting for the longest RTL. Short RTL was independently associated with worse DSS (HR 6.011, 95% CI 1.319–27.397) and PFS (HR 4.689, 95% CI 1.102–19.963) in LBCL treated with R-CHOP-like regimens.ConclusionWe hypothesize that subclones with high global hypomethylation and hypermethylated CpG islands could have advantages in tumor progression, e.g. by inactivating tumor suppressor genes or promoting treatment resistance. Our findings suggest that cases with high global hypomethylation and thus poor prognosis could be candidates for alternative treatment regimens including hypomethylating drugs.

The CpG Island Methylator Phenotype Status in Synchronous and Solitary Primary Colorectal Cancers: Prognosis and Effective Therapeutic Drug Prediction.

Synchronous colorectal cancer (sCRC) is characterized by the occurrence of more than one tumor within six months of detecting the first tumor. Evidence suggests that sCRC might be more common in the serrated neoplasia pathway, marked by the CpG island methylator phenotype (CIMP), than in the chromosomal instability pathway (CIN). An increasing number of studies propose that CIMP could serve as a potential epigenetic predictor or prognostic biomarker of sCRC. Therapeutic drugs already used for treating CIMP-positive colorectal cancers (CRCs) are reviewed and drug selections for sCRC patients are discussed.

Abstract PO1-16-05: Matched multi-analyte and multi-omic liquid biopsy of cell-free DNA, circulating tumor cells and extracellular vesicles in HER2-positive metastatic breast cancer patients

Abstract Background: Liquid biopsy, specifically cell-free DNA (cfDNA) mutation analysis, has recently entered clinical practice for treatment decision-making in hormone receptor (HR)-positive (+)/HER2-negative (-) metastatic breast cancer (mBC) patients. However, we showed that the analysis of multiple liquid biopsy analytes in the course of treatment in this BC subgroup has additive value. Since no multi-analyte studies are currently available for the subgroup of HER2+ mBC patients, we here studied multiple analytes [cfDNA, circulating tumor cells (CTCs) and extracellular vesicles (EVs)] and utilized our multi-omics perspective (CpG island methylation, mutations and mRNA expression) to get insight into their utility in therapy management of HER2+ mBC. Methods: 2x 9 ml blood was drawn from 21 HER2+ (HR+ n=16 and HR- n=5) mBC patients at the time of disease progression and at two consecutive staging time points. CTCs and their mRNA were isolated from 2x5 ml blood by positive immunomagnetic selection targeting EpCAM, EGFR and HER2 (AdnaTest EMT2/StemCell Select/Detect). Plasma of CTC-depleted blood was used for cfDNA isolation, while mRNA from EVs was isolated from 4 ml pre-filtered plasma by affinity-based binding to a spin column (exoRNeasy) using the remaining blood. The mRNA purified from CTCs and EVs by Oligo-dT beads was reverse transcribed, pre-amplified and analyzed by a multi-marker (18 genes) qPCR panel. qPCR data was normalized to CD45 (for CTC analysis) and data of 20 healthy female donor controls to identify BC specific overexpression signals with a specificity of &amp;gt;90% for all targets. Variants in the cfDNA were analyzed with a customized QIAseq Targeted DNA Panel with unique molecular indices (UMIs) and high coverage (mean 22.000x), while CpG island methylation in the cfDNA was analyzed with a customized QIAseq Targeted Methyl Panel with UMIs covering 9786 CpG sites (mean 900x). Consumables: QIAGEN, Germany. Results: In general, substantial differences occurred between the CTC and EV mRNA profiles. Transcripts involved in the PI3K pathway (mTOR, AKT2, PIK3CA) as well as ERBB2 signals were significantly more prevalent in CTCs compared to EVs and mTOR, ERCC1, SRC, AKT2 und PIK3CA CTC overexpression signals were found in &amp;gt;50% of these patients. In contrast, PARP1, AURKA, SRC und ERCC1 signals were significantly more prominent in the EVs and detected in &amp;gt;50% of the patients. ERCC1 signals were present in EVs with highest prevalence whereas ERBB2 signals were only present in CTCs. ERBB3+ CTCs were only detected in patients showing disease progression. Currently, cfDNA mutations as well as cfDNA CpG island methylation are under evaluation and the complete multi-analyte and multi-omic data set as well as their relation to clinical outcome will have been finalized to be presented at the meeting. Conclusion: We successfully established a workflow for parallel isolation of multiple liquid biopsy analytes from a minimized blood volume in HER2+ mBC. Preliminary mRNA profile results for CTCs and EVs show the complementary nature of these two liquid biopsy analytes. Besides the multi-analyte nature of this workflow, the pending results for genomic and epigenomic information will show which one of the analytes, alone or in combination, will help to optimize therapy management in HER2+ mBC patients. Citation Format: Sabine Kasimir-Bauer, Markus Storbeck, Ioanna Andreou, Siegfried hauch, Oliver Hoffmann, Rainer Kimmig, Corinna Keup. Matched multi-analyte and multi-omic liquid biopsy of cell-free DNA, circulating tumor cells and extracellular vesicles in HER2-positive metastatic breast cancer patients [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-16-05.

Abstract PO3-24-05: Cysteine metabolism related ferroptosis sensitivity in trastuzumab resistant HER2 positive breast cancer

Abstract Background Trastuzumab has shown great effectiveness in HER2 positive breast cancer treatment, but about 50% patients would undergo resistance during or after treatment. Although previous research has suggested several potential reasons for trastuzumab resistance, the metabolic reprogramming during resistance formation remains largely unclear. Here we identified aberrant ferroptosis associated cysteine metabolism in trastuzumab resistant HER2 positive breast cancer, which might become a novel target for overcoming resistance. Methods Trastuzumab sensitive HER2 positive breast cancer cell SKBR3 and resistant JITM1 were obtained for transcriptomics, proteomics, metabolomics and epigenomics analysis. Gene silencing was mediated by siRNAs. CUT&amp;Tag was applied to compare H3K4me3 and H3K27me3 binding regions. DNA methylation levels and different methylated regions were evaluated by WGBS-seq. CRISPRi with dCas9-DNMT3A was applied to regulate specific DNA methylation in CpG islands. Lipid ROS was measured by flow cytometry with BODIPY-C11. Results Joint analyses of transcriptomics and proteomics according to ferroptosis pathways revealed downregulated glutathione metabolism, glutamate transmembrane and homocysteine metabolism processes, as well as upregulated fatty acid metabolism and iron metabolism pathways in JIMT1. Metabolomics verified that JIMT1 increased cysteine metabolism and decreased glutathione metabolism. SLC7A11 expression and GSH/GSSG ratio were increased in JIMT1, while no difference was observed in free cysteine. JIMT1 featured significant higher UGC codon usage bias and increased cysteinyl-tRNA synthetase. The abundance of H3K4me3 other than H3K27me3 in SLC7A11 promoter region was found increased in JIMT1, and the 5-mC level of CpG islands in SLC7A11 promoter region was shown decreased. Using dCas9-DNMT3A, the methylation of SLC7A11 promoter was enhanced and SLC7A11 expression was reduced in JIMT1. Inhibition of SLC7A11 by siRNAs, CRISPRi or Erastin all indicated a higher ferroptosis sensitivity in JIMT1. Conclusion Trastuzumab resistant HER2 positive breast cancer features aberrant cysteine metabolism resulting from altered H3K4me3 modification and DNA methylation in SLC7A11 promoter region. This might provide novel targets for further anti-HER2 treatment. Citation Format: Yongmei Yin, Yijia Hua, Ningjun Duan, Chunxiao Sun, Fan Yang. Cysteine metabolism related ferroptosis sensitivity in trastuzumab resistant HER2 positive breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-24-05.

Unraveling the Progression of Colon Cancer Pathogenesis Through Epigenetic Alterations and Genetic Pathways.

In the modern age, colon cancer has attained a widespread status, affecting a considerable number of people.It develops due to the progressive accumulation of genetic and epigenetic alterations. While genetic mutations have been extensively studied in the context of colon cancer, emerging evidence highlights the pivotal role of epigenetic alterations in its pathogenesis. These alterations ultimately result in the transformation of normal colonic epithelium into colon adenocarcinoma.Key mechanisms of epigenetic modifications include DNA methylation, histone modification, and nucleosome positioning. Research findings have linked these modifications to the development, progression, or metastasis of tumors. Through the assessment of the colon cancer epigenome,it has been discovered that practically all colorectal cancers (CRCs) display gene methylation abnormalities and changes in miRNA expression. Advancements in this area indicate that epigenetic modifications will likely be commonly used in the near future to direct the prevention and treatment of CRC. The maintenance of genome stability is essential for preserving cellular integrity. The development of CRC is primarily influenced by the loss of genomic stability, which allows for the emergence of new mutations contributing to tumor characteristics. Although genetic mutations have been extensively researched in the realm of colon cancer, recent evidence underscores the pivotal role of epigenetic changes in its pathogenesis. The following types of genomic instability will be discussed: chromosomal instability, microsatellite instability, CpG island methylation phenotype, and aberrant DNA methylation.

Novel insights into tumorigenesis revealed by molecular analysis of Lynch syndrome cases with multiple colorectal tumors.

Lynch syndrome (LS) is an autosomal dominant multi-organ cancer syndrome with a high lifetime risk of cancer. The number of cumulative colorectal adenomas in LS does not generally exceed ten, and removal of adenomas via routine screening minimizes the cancer burden. However, abnormal phenotypes may mislead initial diagnosis and subsequently cause suboptimal treatment. Currently, there is no standard guide for the care of multiple colorectal adenomas in LS individuals. We aimed to shed insight into the molecular features and reasons for multiplicity of adenomas in LS patients. We applied whole exome sequencing on nine adenomas (ten samples) and three assumed primary carcinomas (five samples) of an LS patient developing the tumors during a 21-year follow-up period. We compared the findings to the tumor profiles of two additional LS cases ascertained through colorectal tumor multiplicity, as well as to ten adenomas and 15 carcinomas from 23 unrelated LS patients with no elevated adenoma burden from the same population. As LS associated cancers can arise via several molecular pathways, we also profiled the tumors for CpG Island Methylator Phenotype (CIMP), and LINE-1 methylation. All tumors were microsatellite unstable (MSI), and MSI was present in several samples derived from normal mucosa as well. Interestingly, frequent frameshift variants in RNF43 were shared among substantial number of the tumors of our primary case and the tumors of LS cases with multiple tumors but almost absent in our control LS cases. The RNF43 variants were completely absent in the normal tissue, indicating tumor-associated mutational hotspots. The RNF43 status correlated with the mutational signature SBS96. Contrary to LS tumors from the reference set with no elevated colorectal tumor burden, the somatic variants occurred significantly more frequently at C>T in the CpG context, irrespective of CIMP or LINE-1 status, potentially indicating other, yet unknown methylation-related mechanisms. There were no signs of somatic mosaicism affecting the MMR genes. Somatic variants in APC and CTNNB1 were unique to each tumor. Frequent somatic RNF43 hot spot variants combined with SBS96 signature and increased tendency to DNA methylation may contribute to tumor multiplicity in LS.

Weighting estimation in the cause-specific Cox regression with partially missing causes of failure.

Complex diseases are often analyzed using disease subtypes classified by multiple biomarkers to study pathogenic heterogeneity. In such molecular pathological epidemiology research, we consider a weighted Cox proportional hazard model to evaluate the effect of exposures on various disease subtypes under competing-risk settings in the presence of partially or completely missing biomarkers. The asymptotic properties of the inverse and augmented inverse probability-weighted estimating equation methods are studied with a general pattern of missing data. Simulation studies have been conducted to demonstrate the double robustness of the estimators. For illustration, we applied this method to examine the association between pack-years of smoking before the age of 30 and the incidence of colorectal cancer subtypes defined by a combination of four tumor molecular biomarkers (statuses of microsatellite instability, CpG island methylator phenotype, BRAF mutation, and KRAS mutation) in the Nurses' Health Study cohort.

Abstract P43: Optimization of DNA Methyltransferase Inhibitors-based Combination to Target the Epigenome of Gastric Cancer

Abstract Aberrant DNA methylation is one of the critical epigenetic modifications that has become a widespread phenotype in solid tumours. Similar to haematological malignancies, many studies have shown that the DNA methylation landscape of solid tumours exhibits a pattern of either hypermethylation of the promoter regions of tumour-suppressive genes in advanced cancer or global hypomethylation in the early stages of carcinogenesis. Additionally, dysregulated DNA methylation also seems to remodel the tumour microenvironment and affect immune cells' response towards immune checkpoint inhibitors. In gastric cancer, comprehensive DNA methylation analyses have demonstrated that gastric tumours carry the CpG island methylation phenotype (CIMP), resulting in extended molecular-based subgroups such as the extremely high-methylation epigenotype (E-HME), HME, and low-methylation epigenotype (LME). Therefore, conventional histology grading and molecular subtyping are insufficient to address the multifaceted epigenome of gastric cancer. As such, current standard-of-care (SOC) therapy is not able to overcome cancer relapse and tumour metastasis. Clearly, this shows the therapeutic potential of using DNA hypomethylating drugs like DNA methyltransferase inhibitors (DNMTi) for solid tumours such as gastric cancer. In our study, we exploited the engineering-based Quadratic Phenotypic Optimization Platform (QPOP) to identify top-ranking DNMTi-based drug combinations across gastric cancer cell lines (GC-CL) and patient-derived organoids (GC-PDO). Based on QPOP analysis, we showed that a non-SOC drug combination, Docetaxel/5-Azacytidine is a frequently top-ranking. Besides, our present findings suggest that CIMP-high gastric cancer cells are more susceptible towards DNMT inhibitors through the specific reduction of DNMT1 levels. As we surveyed the effects of Docetaxel/5-Azacytidine in a CIMP-high GC-CL via Infinium methylation array, we observed robust gene methylation changes that are involved in non-canonical Wnt Signaling and EMT. On the other hand, proteomic analysis revealed a small subset of our panel of GC lines that are positive for the cancer-testis antigen, MageA4. Interestingly, the protein expression of MageA4 in some CIMP-high lines is restored upon treatment with DNMT inhibitors. This evinces that DNMTi can subject epigenetically silenced MageA4 gastric tumours to autologous T-cell receptor therapy. Taken altogether, we plan to further interrogate the mechanisms of DNMT inhibitors in sensitizing CIMP-high gastric tumours towards Taxanes by acting on the non-canonical Wnt pathway and EMT activators. Citation Format: Lissa Hooi, Vivien Koh, Dexter Kai Hao Thng, Jasmine Goh, Lim Jhin Jieh, Lee Rui Xue, Masturah Bte Mohd Abdul Rashid, Toh Tan Boon, Wei Peng Yong, Edward Kai-Hua Chow. Optimization of DNA Methyltransferase Inhibitors-based Combination to Target the Epigenome of Gastric Cancer [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P43.

DNA hypomethylation patterns and their impact on the tumor microenvironment in colorectal cancer.

Recent research underscores the pivotal role of immune checkpoints as biomarkers in colorectal cancer (CRC) therapy, highlighting the dynamics of resistance and response to immune checkpoint inhibitors. The impact of epigenetic alterations in CRC, particularly in relation to immune therapy resistance, is not fully understood. We integrated a comprehensive dataset encompassing TCGA-COAD, TCGA-READ, and multiple GEO series (GSE14333, GSE37892, GSE41258), along with key epigenetic datasets (TCGA-COAD, TCGA-READ, GSE77718). Hierarchical clustering, based on Euclidean distance and Ward's method, was applied to 330 primary tumor samples to identify distinct clusters. The immune microenvironment was assessed using MCPcounter. Machine learning algorithms were employed to predict DNA methylation patterns and their functional enrichment, in addition to transcriptome expression analysis. Genomic mutation profiles and treatment response assessments were also conducted. Our analysis delineated a specific tumor cluster with CpG Island (CGI) methylation, termed the Demethylated Phenotype (DMP). DMP was associated with metabolic pathways such as oxidative phosphorylation, implicating increased ATP production efficiency in mitochondria, which contributes to tumor aggressiveness. Furthermore, DMP showed activation of the Myc target pathway, known for tumor immune suppression, and exhibited downregulation in key immune-related pathways, suggesting a tumor microenvironment characterized by diminished immunity and increased fibroblast infiltration. Six potential therapeutic agents-lapatinib, RDEA119, WH.4.023, MG.132, PD.0325901, and AZ628-were identified as effective for the DMP subtype. This study unveils a novel epigenetic phenotype in CRC linked to resistance against immune checkpoint inhibitors, presenting a significant step toward personalized medicine by suggesting epigenetic classifications as a means to identify ideal candidates for immunotherapy in CRC. Our findings also highlight potential therapeutic agents for the DMP subtype, offering new avenues for tailored CRC treatment strategies.

Abstract 4410: The CpG island methylator phenotype is closely associated with prognosis in patients with oral squamous cell carcinomas

Abstract Few abnormalities are known to be closely associated with prognosis in OSCC cases. In this study, genomic abnormalities of cancer-related genes and epigenomic abnormalities of genes methylation-silenced in OSCCs were analyzed to identify abnormalities associated with prognosis. Thirty-nine OSCC cases and their surgical materials were analyzed for the presence of mutations using next-generation sequencing. Methylation status of promoter CpG islands of five genes was analyzed by methylation-specific PCR. Mutations in CRG, TP53 and PIK3CA were observed in 64.1%, 38.5%, and 33.3%, respectively, of the cases. However, no significant association between mutation status and overall survival (OS) was observed. Aberrant methylation of CMTM3, NKX2-3, RBP4, EGFLAM, and MAP6 were observed in 51.3%, 41.0%, 66.7%, 38.5%, and 41.0%, respectively. Methylation of NKX2-3, EGFLAM, and MAP6 were significantly associated with OS in OSCC cases (p=0.006, 0.006, and 0.012, respectively). Cases with methylation of CMTM3 and RBP4 showed a trend toward poor prognosis (p=0.052 and 0.059, respectively). Methylation profiles of the five genes indicated the presence of the CpG island methylator phenotype (CIMP), and the CIMP-positive cases showed poor prognosis (HR=20.5, p&amp;lt;0.001). In conclusion, CIMP was closely associated with the prognosis of OSCC cases. Citation Format: Masanobu Abe, Satoshi Yamashita, Toshikazu Ushijima, Kazuto Hoshi. The CpG island methylator phenotype is closely associated with prognosis in patients with oral squamous cell carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4410.

Abstract 2866: NAPRTsilencing in rhabdomyosarcoma confers therapeutic vulnerabilities to NAD+depletion

Abstract Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma among children, and new treatments are needed to improve survival. Cells primarily synthesize nicotinamide adenine dinucleotide (NAD) through the Preiss-Handler and Salvage pathways driven by nicotinic acid phosphoribosyltransferase (NAPRT) and nicotinamide phosphoribosyltransferase (NAMPT), respectively. NAMPT inhibitors (NAMPTi) have been tested in clinical trials, but their efficacy has been limited by (a) lack of appropriate biomarkers for patient selection, and (b) significant dose-limiting toxicities (DLTs), including myelosuppression and retinal toxicity, although the latter has been described only in preclinical studies. Previous work from our group indicates that loss of NAPRT expression by tumor-specific promoter CpG island methylation may provide a novel biomarker for sensitivity to NAMPTi in a variety of tumors. Here, we sought to determine if RMS harbors NAPRT silencing that confers a synthetic lethal interaction with NAMPTi. We probed methylation data within the Cancer Dependency Map (DepMap) and found a high frequency of NAPRT gene promoter hypermethylation in RMS models. Predicted NAPRT silencing was confirmed at the protein level by western blot in a panel of RMS cell lines. In vitro growth delay assays demonstrated that NAPRT-silenced cells are exquisitely sensitive to NAMPTi even in the presence of nicotinic acid (NA) supplementation, while NAPRT-expressing cells were completely rescued. This sensitivity to NAMPTi was further mirrored by changes in NAD levels. In vivo, NAMPTi induced significant tumor regression in a NAPRT-silenced RMS xenograft model. To further establish the translational relevance, NAPRT protein expression in a TMA of 51 human RMS tumors was assessed via IHC staining using a newly validated NAPRT monoclonal antibody (4A5D7). Based on a semi-quantitative scoring system accounting for the percentage of positive tumor cells and staining intensity, 28% of RMS samples demonstrated loss of NAPRT protein expression. Overall, these data suggest that NAPRT loss may serve as a therapeutic target in RMS by inducing a synthetic lethal interaction with NAD+ depleting agents. Future studies in patient-derived xenograft (PDX) models will determine the optimal cut-off of NAPRT expression that portends NAMPTi sensitivity, paving the way for the development of biomarker-driven clinical trials in RMS. Ongoing studies will also evaluate the efficacy of combining NAMPTi with existing cytotoxic and DNA damaging chemotherapy in these difficult-to-treat tumors. Citation Format: Sophia J. Zhao, Katelyn J. Noronha, Prateek Bhardwaj, Karlie N. Lucas, Ranjini K. Sundaram, Collin D. Heer, Raffaella Morotti, Josh Spurrier, Mitch Raponi, Ranjit S. Bindra, Juan C. Vasquez. NAPRTsilencing in rhabdomyosarcoma confers therapeutic vulnerabilities to NAD+depletion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2866.

Abstract 3241: Elevated H3K27 trimethylation mediates adaptation to DNA demethylation in BRAFV600E-mutated colorectal cancer

Abstract Background: BRAFV600E in colorectal cancer (CRC) defines a unique subgroup of patients with poor prognosis and scarcely benefit from BRAF inhibitor-based treatment. Interestingly, BRAFV600E tumors demonstrated profound CpG island methylator phenotype (CIMP), suggesting extensive epigenetic dysregulation. Thus, we investigated epigenomic regulations in this subset of tumors with treatment-induced DNA demethylation. Methods: We treated six cell lines and two patient-derived xenograft (PDX) models of BRAFV600E CRC with vehicle, 5-azacitidine (DNMTi), vemurafenib (BRAFi), and combination treatment. PDX tumors treated with either vehicle or 5-azacitidine were collected on day 21 and sequenced for DNA methylation (Infinium HumanMethylation 450k BeadChip array), histone marks (ChIP-seq), chromatin accessibility (ATAC-seq), and gene expression (RNA-seq). Comprehensive post-translational modifications (PTMs) in histones with DNMTi or BRAFi treatment were assessed by mass spectrometry (MS). The combinational effect of 5-azacitidine with EZH2 or RNF2 inhibitors (Polycomb repressive complex activity (PRC) inhibition) was assessed through cell viability assays and colony formation assays in BRAFV600E CRC cell lines. Results: Analysis of TCGA COAD/READ tumors demonstrated a higher DNA methylation profile in BRAFV600E tumors than wild-type tumors even among the CIMP population (FDR &amp;lt; 1e-4). The combination of DNMTi with BRAFi showed selective additivity in vitro; however, did not improve efficacies in PDXs. The 5-azacitidine treatment induced profound demethylation in PDXs compared to control samples, predominantly attributed to promoter regions (48%). However, DNA demethylation failed to reactivate classic CIMP markers or tumor suppressor genes in CRC (e.g., CDKN2A, MGMT). Interestingly, demethylated genomic regions gained H3K27 trimethylation, repressive histone mark, in azacitidine-treated samples compared to control (9,431 vs 5,147 peaks respectively). We validated the elevation of H3K27 trimethylation as well as additional histone PTMs following DNMTi in MS-based histone analysis. Finally, combining a demethylating agent with EZH2i or RNF2i, which either deplete H3K27 trimethylation or block the PRC activity respectively, has demonstrated additive growth inhibitory effects in BRAFV600E CRC cell lines (p &amp;lt;0.05). Conclusions: This study unveiled intriguing aspects of demethylation agent treatment, which led to the compensatory engagement of other epigenomic markers, particularly repressive histone marks, resulting in limited efficacies to azacitidine treatment in preclinical BRAFV600E CRC models. This finding suggests the existence of adaptive interactions between epigenetic modifiers and potential clinical strategies in combinational epigenetic therapeutics. Citation Format: Hey Min Lee, Ajay Kumar Saw, Van Karlyle Morris, Anand Kamal Singh, Stefania Napolitano, Alexey Sorokin, Preeti Kanikarla Marie, Oluwadara Coker, Oscar Eduardo Villarreal, Nazanin Esmaeili Anvar, Kunal Rai, Scott Kopetz. Elevated H3K27 trimethylation mediates adaptation to DNA demethylation in BRAFV600E-mutated colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3241.

Epigenetic MLH1 silencing concurs with mismatch repair deficiency in sporadic, naturally occurring colorectal cancer in rhesus macaques

BackgroundNaturally occurring colorectal cancers (CRC) in rhesus macaques share many features with their human counterparts and are useful models for cancer immunotherapy; but mechanistic data are lacking regarding the comparative molecular pathogenesis of these cancers.MethodsWe conducted state-of-the-art imaging including CT and PET, clinical assessments, and pathological review of 24 rhesus macaques with naturally occurring CRC. Additionally, we molecularly characterized these tumors utilizing immunohistochemistry (IHC), microsatellite instability assays, DNAseq, transcriptomics, and developed a DNA methylation-specific qPCR assay for MLH1, CACNA1G, CDKN2A, CRABP1, and NEUROG1, human markers for CpG island methylator phenotype (CIMP). We furthermore employed Monte-Carlo simulations to in-silico model alterations in DNA topology in transcription-factor binding site-rich promoter regions upon experimentally demonstrated DNA methylation.ResultsSimilar cancer histology, progression patterns, and co-morbidities could be observed in rhesus as reported for human CRC patients. IHC identified loss of MLH1 and PMS2 in all cases, with functional microsatellite instability. DNA sequencing revealed the close genetic relatedness to human CRCs, including a similar mutational signature, chromosomal instability, and functionally-relevant mutations affecting KRAS (G12D), TP53 (R175H, R273*), APC, AMER1, ALK, and ARID1A. Interestingly, MLH1 mutations were rarely identified on a somatic or germline level. Transcriptomics not only corroborated the similarities of rhesus and human CRCs, but also demonstrated the significant downregulation of MLH1 but not MSH2, MSH6, or PMS2 in rhesus CRCs. Methylation-specific qPCR suggested CIMP-positivity in 9/16 rhesus CRCs, but all 16/16 exhibited significant MLH1 promoter hypermethylation. DNA hypermethylation was modelled to affect DNA topology, particularly propeller twist and roll profiles. Modelling the DNA topology of a transcription factor binding motif (TFAP2A) in the MLH1 promoter that overlapped with a methylation-specific probe, we observed significant differences in DNA topology upon experimentally shown DNA methylation. This suggests a role of transcription factor binding interference in epigenetic silencing of MLH1 in rhesus CRCs.ConclusionsThese data indicate that epigenetic silencing suppresses MLH1 transcription, induces the loss of MLH1 protein, abrogates mismatch repair, and drives genomic instability in naturally occurring CRC in rhesus macaques. We consider this spontaneous, uninduced CRC in immunocompetent, treatment-naïve rhesus macaques to be a uniquely informative model for human CRC.Graphical abstract