Hypermethylation Of Tumor Suppressor Genes Research Articles

Abstract 1484: 5-Aza-4'-thio-2'-deoxycytidine induces C to G transversions in a specific trinucleotide context and leads to acute lymphoid leukemia

Abstract Myelodysplastic syndromes (MDS) are a diverse group of blood cancers characterized by peripheral blood cytopenias and dysplastic blood cell morphology. Up to 40% of patients with high risk MDS will transform to acute myeloid leukemia (AML), an aggressive blood cancer with significant morbidity and an overall 5-year survival of less than 35%. Both MDS and AML have been linked to hypermethylation of cytosine residues. Hypermethylation in the 5’ regulatory region can result in epigenetic downregulation of gene expression, and it has been speculated that hypermethylation of tumor suppressor genes can contribute to malignant transformation. DNA methyltransferase inhibitors (DNMTi), most commonly cytidine analogs, are compounds that are used clinically to decrease 5’-cytosine methylation, with the aim of inducing the re-expression of tumor suppressor genes resulting in a halt of tumor progression. 5-Aza-4’-thio-2’-deoxycytidine (Aza-TdCyd or ATC) is a cytidine analog that incorporates an aza modification (nitrogen in place of carbon) of the cytosine base and a thioether modification of the deoxyribose sugar. ATC is a promising new DNMT1i that has demonstrated efficacy in preclinical studies of solid tumors. Based on these prior studies, we sought to characterize the potential therapeutic effect of ATC in MDS.Mice that express a NUP98::HOXD13 (NHD13) transgene develop an MDS that closely recapitulates all of the key features of human MDS, including pancytopenia, dysplastic morphology, and transformation to AML. We transplanted NHD13 and wild-type (WT) hematopoietic stem and progenitor cells into WT recipients, and used this model of MDS to investigate the therapeutic potential of ATC. We found that ATC treatment led to both T and B lineage lymphoid leukemia (ALL) originating from the NHD13 cells. Surprisingly, ATC treatment also led to T and B lineage ALL in the WT cells, and further experiments demonstrated that non-transplanted, healthy WT mice developed T and B cell ALL after treatment with ATC. In sum, all mice that received at least 8 cycles of ATC therapy at the 1.0 mg/kg dose developed leukemia. Whole exome sequencing of tumors revealed thousands of acquired mutations, almost all of which were C>G transversions in a previously unrecognized, specific 5’-NCG-3’ context. These mutations involved a number of genes well-known to be involved in human lymphoid leukemia, such as Notch1, Pten, Pax5, Trp53, and Nf1. Treatment of the human U937 and CEM cell lines in vitro showed thousands of acquired C>G transversions in a similar context. Taken together, these findings demonstrate that ATC can be a potent mutagen in human and mouse cells, both in vitro and in vivo. Citation Format: Ryan Matthew Bertoli, Yang Jo Chung, Michael Difilippantonio, Anthony Wokasch, Madison R. Marasco, Haley Klimaszewski, Susannah Garber, Yuelin Jack Zhu, Robert L. Walker, Dengchao Cao, James Doroshow, Paul Meltzer, Peter D. Aplan. 5-Aza-4'-thio-2'-deoxycytidine induces C to G transversions in a specific trinucleotide context and leads to acute lymphoid leukemia [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 1484.

Abstract 7003: Microfluidic platform for DNA methylation profiling towards early detection of ovarian cancer

Abstract We present a microfluidic platform for molecule-by-molecule detection of heterogeneous epigenetic patterns of rare tumor-derived DNA by highly parallelized digital melt. The platform digitizes DNA molecules in a 4-module, 40,000 1-nL array microfluidic device and observes sequence-dependent fluorescence changes during temperature ramping. The platform is applied to quantify DNA methylation heterogeneity of a biomarker panel within Pap specimens in a small cohort of healthy and ovarian cancer patients, and demonstrates an accuracy of 0.9. DNA hypermethylation of tumor-suppressor genes is an epigenetic phenomena that occurs early in tumorigenesis. Variable methylation patterns are observed in ovarian cancer precursor tissue, and trace amounts of ovarian-tumor-derived DNA can be found in Pap specimens, albeit at low frequencies (<0.01%). This suggests that methylation profiles of DNA extracted from routinely-obtained Pap specimens could contain early cancer biomarkers. However, due to the scarcity of these molecules, current techniques, such as sequencing and digital PCR (dPCR), have technical limitations precluding their ability to profile methylation patterns reliably. Sequencing, although comprehensive, has limited sensitivity, undermining its utility for routine detection of fractions below 0.1%. dPCR achieves high sensitivity, but is limited to known sequences, thus cannot provide comprehensive analysis of molecular heterogeneity. To overcome these limitations, we developed a technique called digital high resolution melt (dHRM), a thermodynamics-based approach to detecting molecular variability by observing the sequence-specific release of a DNA-intercalating dye under a thermal ramp. dHRM can discriminate methylation patterns on CpG-by-CpG basis and detect methylated epialleles at frequencies as low as 0.00005%. Here, DNA from Pap specimens was loaded on a microfluidic chip that digitizes rare target molecules into individual reaction chambers. A thermal-optical platform was developed to perform parallelized dPCR and dHRM. Methylation patterns of a biomarker panel were analyzed to produce a probability score that the Pap specimen contained tumor-derived DNA. 12 healthy and 12 ovarian cancer Pap specimens were assessed for methylation heterogeneity of 9 genes. Optimal methylation density thresholds were determined, and data above each threshold was combined into a single score for each possible combination of biomarkers. In this small cohort, methylation heterogeneity analysis of just 2 loci produced an area under the receiver-operator characteristics curve (AUC) of 0.9. This highly sensitive methylation profiling technology demonstrates promising utility towards early cancer detection. Future work aims to expand the testing cohort for ovarian cancer detection and permit further studies on the impact of methylation heterogeneity on early cancer development. Citation Format: Chrissy O'Hersey, Yang Zhao, Thomas R. Pisanic, Tian-Li Wang, Ie-Ming Shih, Tza-Huei Jeff Wang. Microfluidic platform for DNA methylation profiling towards early detection of ovarian 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 7003.

Clinical Significance of Frequently Down-Regulated Phosphatidylethanolamine-Binding Protein-1 in Gallbladder Cancer.

Promoter hypermethylation of tumor suppressor genes has been demonstrated to be one of the major mechanisms of their epigenetic regulation in various reports. We have studied the promoter methylation status of PEBP1 and evaluated its correlation with gallbladder carcinogenesis. PEBP1, an endogenous inhibitor of Raf/MEK/ERK signaling pathway, is a tumor suppressor gene. We aimed to study the expression profile of PEBP1 and understand the mechanism and significance of its deregulation in gallbladder cancer. PEBP1 expression analysis and its promoter methylation status were investigated in 77 gallbladder carcinoma (GBC) and tissue biopsies from 28 patients of gallstone disease by RT-PCR and MS-PCR, respectively. Our results of the mRNA expression profiling demonstrate that PEBP1 is down-regulated in 62.3% (48/77), while 31.2% (24/77) of the gallbladder cancer biopsies show no significant change and 6.5% (5/77) show up-regulated expression compared to tissue samples of gallstone diseases. In GBC, 48.1% (N = 37) GBC biopsy samples exhibited significantly heterozygous promoter hypermethylation compared to tissue samples from gallstone diseases which show promoter hypermethylation in 3 (10.7%) samples only. In gallbladder cancer, the PEBP1 methylation is significantly associated with lymph node metastasis and shorter period of survival. PEBP1 is frequently down-regulated and hypermethylated in gallbladder cancer and its promoter hypermethylation is a frequent and early inactivating mechanism in GBC.

Comparing the Costs and Diagnostic Outcomes of Replacing Cytology with the QIAsure DNA Methylation Test as a Triage within HPV Primary Cervical Cancer Screening in The Netherlands.

Detecting hypermethylation of tumour suppressor genes could provide an alternative to liquid-based cytology (LBC) triage within HPV primary cervical screening. The impact of using the QIAsure® FAM19A4/mir124-2 DNA Methylation Test (QIAGEN, N.V, Hilden, Germany) on CIN3+ diagnoses, retention, unnecessary colposcopies, and programme costs is unknown. A decision-tree model was developed to compare LBC with the QIAsure Methylation testing to guide colposcopy referral. Incorporating clinician- and self-sampling pathways the model was informed by the Dutch cervical cancer screening programme, published studies, and manufacturer data. Clinical and cost outcomes were assessed using two scenarios for DNA methylation testing and LBC relative performance. Sensitivity analyses (deterministic and probabilistic) were performed to assess model and parameter uncertainty. A range of self-sampling uptake was assessed in scenario analyses. For the screening cohort (n = 807,269) where 22.1% self-sampled, the number of unnecessary colposcopies and CIN3+ diagnoses varied according to the relative performance of methylation testing and LBC. Irrespective of relative performance, the cost per complete screen was lower and fewer people were lost to follow-up when using DNA methylation testing. The results indicate that, within an HPV primary screening programme that incorporates self-sampling, using the QIAsure Methylation Test for triage reduces the cost per screen compared to LBC.

Helicobacter pylori-associated Chronic Atrophic Gastritis and Progression of Gastric Carcinogenesis.

Chronic inflammation due to a Helicobacter pylori (H. pylori) infection is a representative cause of gastric cancer that can promote gastric carcinogenesis by abnormally activating immune cells and increasing the inflammatory cytokines levels. H. pylori infections directly cause DNA double-strand breaks in gastric epithelial cells and genetic damage by increasing the enzymatic activity of cytidine deaminase. Eventually, gastric cancer is induced through dysplasia. Hypermethylation of tumor suppressor genes is an important cause of gastric cancer because of a H. pylori infection. In addition, the changes in gastric microbiota and the mucosal inflammatory changes associated with a co-infection with the Epstein-Barr virus are associated with gastric cancer development. DNA damage induced by H. pylori and the subsequent responses of gastric stem cells have implications for gastric carcinogenesis. Although the pathogenesis of H. pylori has been established, many uncertainties remain, requiring more study.

CRISPR/dCAS9-mediated DNA demethylation screen identifies functional epigenetic determinants of colorectal cancer

BackgroundPromoter hypermethylation of tumour suppressor genes is frequently observed during the malignant transformation of colorectal cancer (CRC). However, whether this epigenetic mechanism is functional in cancer or is a mere consequence of the carcinogenic process remains to be elucidated.ResultsIn this work, we performed an integrative multi-omic approach to identify gene candidates with strong correlations between DNA methylation and gene expression in human CRC samples and a set of 8 colon cancer cell lines. As a proof of concept, we combined recent CRISPR-Cas9 epigenome editing tools (dCas9-TET1, dCas9-TET-IM) with a customized arrayed gRNA library to modulate the DNA methylation status of 56 promoters previously linked with strong epigenetic repression in CRC, and we monitored the potential functional consequences of this DNA methylation loss by means of a high-content cell proliferation screen. Overall, the epigenetic modulation of most of these DNA methylated regions had a mild impact on the reactivation of gene expression and on the viability of cancer cells. Interestingly, we found that epigenetic reactivation of RSPO2 in the tumour context was associated with a significant impairment in cell proliferation in p53−/− cancer cell lines, and further validation with human samples demonstrated that the epigenetic silencing of RSPO2 is a mid-late event in the adenoma to carcinoma sequence.ConclusionsThese results highlight the potential role of DNA methylation as a driver mechanism of CRC and paves the way for the identification of novel therapeutic windows based on the epigenetic reactivation of certain tumour suppressor genes.

Hybridization-based CpG methylation level detection using methyl-CpG-binding domain–fused luciferase

Hypermethylation of tumor-suppressor genes and global hypomethylation, which is related to methylation level at the retroelement, have been recognized as features of the cancer genome. In this study, we developed a hybridization-based CpG methylation level detection method using methyl-CpG-binding domain-fused firefly luciferase (MBD-Fluc). In this method, methylated probe oligonucleotides were used to capture target oligonucleotides. Fully methylated and hemimethylated double-stranded DNA (dsDNA) was formed by hybridization of the methylated captured oligonucleotides with methylated or unmethylated target oligonucleotides, respectively. MBD-Fluc specifically binds to fully methylated dsDNA but not to hemimethylated dsDNA; therefore, methylated target oligonucleotides can be detected by measuring the luciferase activity of the bound MBD-Fluc. Using the corresponding methylated probe oligonucleotides, the CpG methylation levels of SEPT9, BRCA1, and long interspersed nuclear element-1 (LINE-1) oligonucleotides were quantified. Moreover, we demonstrated that the emission detection signal was not affected by the methylation state of the overhang region of the target oligonucleotide, which was not hybridized to the probe oligonucleotide, indicating that methylated CpG of the target region could be accurately detected. Unmethylated-CpG-binding domain-fused luciferases and 5-hydroxymethyl-CpG-binding domain-fused luciferases have been constructed, suggesting that other modified bases can be detected by the same platform.

Universal Design of Luciferase Fusion Proteins for Epigenetic Modifications Detection Based on Bioluminescence Resonance Energy Transfer.

Global hypomethylation and promoter hypermethylation of tumor-suppressor genes are the hallmarks of cancer. We previously reported a global DNA methylation level sensing system based on dual-color bioluminescence resonance energy transfer (BRET) using methyl-CpG binding domain (MBD)-fused firefly luciferase (Fluc) and unmethyl-CpG binding domain (CXXC)-fused Oplophorus luciferase (Oluc). Moreover, BRET-based hydroxymethylation and hemi-methylation level sensing systems have been developed using hydroxymethyl-CpG and hemi-methyl-CpG binding domain-fused Fluc. These studies suggest that target epigenetic modifications can be simultaneously quantified using target-modification-binding protein-fused luciferases. In this study, we focused on the SnoopTag (SnT)/SnoopCatcher (SnC) protein ligation system to establish a universal design for fusion protein construction for any combination. SnT spontaneously forms an isopeptide bond with SnC; therefore, any kind of fusion protein would be constructed by the SnT/SnC system. To establish the proof of concept, MBD-SnT, CXXC-SnT, and SnC-Oluc were prepared and ligated MBD-SnT or CXXC-SnT to SnC-Oluc. The ligation products of MBD-SnT-SnC-Oluc and CXXC-SnT-SnC-Oluc showed luciferase activity and specific binding activity to methyl-CpG and unmethyl-CpG, respectively. The BRET signal using MBD-SnT-SnC-Oluc and CXXC-SnT-SnC-Oluc increased the amount of methyl-CpG and unmethyl-CpG in genomic DNA, respectively. There was a significant negative correlation between the BRET signals; therefore, the global DNA methylation level was quantified using the BRET signals (R2 = 0.99, and R.S.D. <3.5%). These results indicate that the SnT/SnC protein ligation system can be utilized to construct target modification-binding protein-fused luciferases in any combination that detects target modifications in genomic DNA based on BRET.

An Optimized CoBRA Method for the Microfluidic Electrophoresis Detection of Breast Cancer Associated RASSF1 Methylation.

Although breast cancer screening assays exist, many are inaccessible and have high turnaround times, leaving a significant need for better alternatives. Hypermethylation of tumor suppressor genes is a common epigenetic marker of breast cancer. Methylation tends to occur most frequently in the promoter and first exon regions of genes. Preliminary screening tests are crucial for informing patients whether they should pursue more involved testing. We selected RASSF1, previously demonstrated to be aberrantly methylated in liquid biopsies from breast cancer patients, as our gene of interest. Using CoBRA as our method for methylation quantification, we designed unique primer sets that amplify a portion of the CpG island spanning the 5' end of the RASSF1 first exon. We integrated the CoBRA approach with a microfluidics-based electrophoresis quantification system (LabChip) and optimized the assay such that insightful results could be obtained without post-PCR purification or concentration, two steps traditionally included in CoBRA assays. Circumventing these steps resulted in a decreased turnaround time and mitigated the laboratory machinery and reagent requirements. Our streamlined technique has an estimated limit of detection of 9.1 ng/μL of input DNA and was able to quantify methylation with an average error of 4.3%.

Epigenetic methylation changes: implication as biomarkers in oral and maxillofacial area cancers.

Squamous cell carcinoma (SCC) is the most frequent cancer of the head and neck area in the oral cavity. Epigenetic alterations in oral and maxillofacial area cancers are urgently needed to be investigated, as the observed changes might have crucial diagnostic value for personalized medicine. Our study aimed to identify the most frequently hypermethylated tumor suppressor gene promoters in OSCC, followed by correlation analysis with the patients' survival. We evaluated the methylation status of the promoters in a panel of 22 tumor suppressor genes in Romanian (n=9) and Bulgarian (n=12) patient groups suffering from oral and maxillofacial area cancers. The extracted DNA was further digested through EpiTect Methyl II PCR Array System containing methylation-sensitive and methylation-dependent restriction enzymes, followed by specific amplification of the products obtained by qPCR and data analysis using the online platform provided by the producer. Different methylation patterns were observed in the tumor suppressor genes' promoters. Among them, the methylation profile of Cccnd2, Chd1, Cdh13, Cdkn1c, Neurog1, Gstp1, and Runx3 genes further correlated with overall survival rates. Our data emphasize that epigenetic alterations are responsible for the clinical heterogeneity of oral and maxillofacial area cancers and significantly impact on patient survival. Additional investigation on a larger patient cohort should validate these potential biomarkers.

Targeting DNA Methylation in Leukemia, Myelodysplastic Syndrome, and Lymphoma: A Potential Diagnostic, Prognostic, and Therapeutic Tool.

DNA methylation represents a crucial mechanism of epigenetic regulation in hematologic malignancies. The methylation process is controlled by specific DNA methyl transferases and other regulators, which are often affected by genetic alterations. Global hypomethylation and hypermethylation of tumor suppressor genes are associated with hematologic cancer development and progression. Several epi-drugs have been successfully implicated in the treatment of hematologic malignancies, including the hypomethylating agents (HMAs) decitabine and azacytidine. However, combinations with other treatment modalities and the discovery of new molecules are still the subject of research to increase sensitivity to anti-cancer therapies and improve patient outcomes. In this review, we summarized the main functions of DNA methylation regulators and genetic events leading to changes in methylation landscapes. We provide current knowledge about target genes with aberrant methylation levels in leukemias, myelodysplastic syndromes, and malignant lymphomas. Moreover, we provide an overview of the clinical trials, focused mainly on the combined therapy of HMAs with other treatments and its impact on adverse events, treatment efficacy, and survival rates among hematologic cancer patients. In the era of precision medicine, a transition from genes to their regulation opens up the possibility of an epigenetic-based approach as a diagnostic, prognostic, and therapeutic tool.

DNA Methylation: From Cancer Biology to Clinical Perspectives.

DNA methylation plays an important role in the silence of tissue-specific genes to prevent them from being expressed in the wrong tissue. Aberrant DNA methylation (genome-wide hypomethylation and site-specific hypermethylation) are observed in many types of cancer. DNA methylation patterns are established and maintained through the combined actions of methyltransferase and demethylase, such as DNA methyltransferase (DNMT)-1, DNMT-3, and ten-eleven translocation (TET) family enzymes. It is well known that the process of tumor evolution is complicated with different hallmarks. Early findings put forward the model that focal hypermethylation of tumor suppressor genes (TSG) could straightly trigger transcriptional silencing and malignant transformation, whereas varying levels of DNA methylation also occur at other sites and can differently regulate gene expression and biological processes. The interplay of tumor and immune cells in the tumor microenvironment is complex. Understanding the role of DNA methylation in cancer immunity is critical to better navigate epigenetic agents. Furthermore, a greater understanding of the interaction of DNA methylation with tumor metabolic reprogramming would create a bright avenue for pharmacologic managements of malignancies. In this review, we will describe the molecular mechanisms of DNA methylation abnormalities in cancer biology, introduce the roles of DNA methylation patterns on cancer-immunity cycle and metabolic reprogramming, summarize modulators that are used in targeting DNA remodeling, and highlight the importance of combining epigenome-targeting drugs with other cancer therapies.

Demethylation of CADM1 and SOCS1 using capsaicin in cervical cancer cell line.

Cervical cancer is one of the leading causes of women's mortality in developing countries. The prevalence of cervical cancer is higher in developing countries like India and continents like Africa. Hyper-methylation of tumor suppressor genes through human papillomavirus (HPV) infection is known to be one of the major causes of cervical cancer. The promoter hypermethylation of the cell adhesion molecule 1 (CADM1) and suppressor of cytokine signalling (SOCS1) genes due to DNMT1 overexpression leads to their epigenetic silencing followed by gene repression causing cervical cancer. In silico study on the inhibition effect of capsaicin on DNMT1 was simulated by different servers. The binding energy was observed to be -7.8 kcal/mol. In vitro studies on the effect of capsaicin on aberrant methylation of CADM1 and SOCS1 were performed on the adenocarcinoma cervical cancer cell line, HeLa. The IC50 of capsaicin was observed to be 160 μM through crystal violet assay. DNA methylation of the CADM1 and SOCS1 was analyzed by methylation-specific PCR along with their reversal using capsaicin (20 μM) by treating the cells for 72 h and 6 days. In silico results suggested that capsaicin has an inhibitory effect on DNMT1, which regulates DNA methylation leading to the hypermethylation of CADM1 and SOCS1 genes. The in vitro studies suggested that hypermethylation leads to the inhibition of CADM1 and SOCS1 expression, which could be reversed using capsaicin with visible changes in methylation-specific and unmethylation-specific bands in MS-PCR, respectively. The present study shows the reversal of methylation of CADM1 and SOCS1 after 72 h which showed a further increase in case of 6 days of treatment using 20 μM capsaicin, which makes capsaicin a potent candidate for causing demethylation of CADM1 and SOCS1 genes that may lead to the reactivation of the downregulated gene.

DNA Methylation-Based Burkitt Lymphoma Epitypes Have Distinct Molecular and Clinical Features

Introduction: Genome-wide hypomethylation coupled with hypermethylation of tumor suppressor genes is a hallmark of many cancers. Consistent changes in DNA methylation patterns are also a feature of immune cells as they undergo terminal differentiation and maturation. In previous studies, it has been shown that malignant B cells harbor a combination of de novo methylation while retaining some epigenetic features characteristic of their cell of origin. Although information on the genetic features of Burkitt Lymphoma (BL) is expanding, limited information exists on the methylation landscape and its role in pathogenesis. We sought to identify consistent DNA methylation changes and their interplay between BL pathogenesis and other features such as EBV status and somatic mutations. We performed an integrative analysis using comprehensive genome-wide CpG methylation profiling, whole genome sequencing, and gene expression analysis. Methods: Samples were collected as part of the Burkitt Lymphoma Genome Sequencing Project (BLGSP). DNA from 218 primary BL tumors (154 pediatric (111 EBV+), 64 adult (19 EBV+)), 13 BL cell lines (6 EBV+), and 6 normal centroblast (CB) samples were profiled using the Illumina EPIC array. DNA and RNA from biopsies and cell lines underwent whole genome and RNA sequencing to enable the correlation of DNA methylation status with gene expression and genomic alterations. Nine of the BL samples were separately subjected to whole genome sequencing using the PromethION (Oxford Nanopore) to infer genome-wide methylation landscape. Publicly available data from 1595 B cell samples representing both normal subpopulations and tumor subtypes were used for further comparison. To search for methylation patterns within BL, beta values from the 10000 most variable CpGs were used as features for non-negative matrix factorization (NMF) clustering. Results: Clustering revealed two distinct epigenetic subgroups (epitypes) within BL, which we have named hyperBL (cluster1) and hypoBL (cluster2). We found pediatric and adult tumors to be proportionally split across the two epitypes; however, we noted an over-representation of EBV+ tumors in hyperBL. PCA analysis comparing BL with the additional B-cell samples revealed a separation between the epitypes, with hyperBL projecting farther along the trajectory of B cell differentiation, suggestive of a more terminally differentiated B-cell. Investigating the differential methylation between epitypes revealed hyperBL to be associated with greater hypermethylation compared to hypoBL and CBs. The hypermethylated CpGs in hyperBL mainly affected promoter regions within CpG islands while hypomethylated CpGs were mostly intergenic or in open seas. Using PromethION data to allow for genome-wide analyses, we found hypermethylated regions of hyperBL were enriched for binding motifs for several transcription factors involved in B cell development including: IRF4, PAX5, YY1, BCL6, SP1, STAT3, and KLF9. We used mitotic clock estimates based on DNA methylation (epiCMIT) and found hyperBL had significantly elevated values, suggesting a greater proliferative history of these tumors. To further establish a molecular basis for these epitypes, we searched for gene expression differences and distinct driver mutation patterns. The genomes of hyperBL tumors harbored an overall greater mutation burden and significantly higher mutation burden within regions affected by aberrant somatic hypermutation (aSHM), which was associated with a higher frequency of mutations attributed to the SBS9 mutational signature. Gene expression differences indicate hypoBL tumors have upregulation of IRF4 induced pathways. To determine the clinical relevance of the unique epigenetic clusters we conducted survival analyses and found hyperBL samples to have significantly inferior progression free survival. Conclusion: This work identifies novel epigenetic subgroups within BL with characteristic genetic differences between them. HyperBL is associated with hypermethylation, a higher mutation burden, elevated rates of aSHM, and inferior PFS. These results establish a subset of patients with distinct epigenetic, molecular and clinical features and further elucidates key mechanisms underlying BL pathogenesis. Together these data highlight the need and potential for strategies that exploit these epigenetic changes as a therapeutic target. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

DOK7 CpG hypermethylation in blood leukocytes as an epigenetic biomarker for acquired tamoxifen resistant in breast cancer.

Breast cancer (BC) is among the most common cause of cancer 10.4% and one of the leading causes of death among 20-50 years old women in the world. Tamoxifen drug is the first line therapy for BC however tamoxifen resistance (TR) has shown in 30-50% of cases that may face BC recurrence. Hence, TR early detection reduces BC recurrence and fatalities. The epigenetic alteration that happens by hypermethylation of tumor suppressor genes and hypomethylation of oncogenes has been suggested to be useful in early cancer or drug resistance diagnosis. This is the first study to investigate DOK7 CpG hypermethylation in blood leukocytes of 31 TR (ER+) BC compared to 29 tamoxifen sensitive BC to evaluate DOK7 as a potential TR biomarker. DNA was extracted from blood samples of all participants and MSRE-PCR and real-time PCR were used for quantification of CpG methylation alterations. The means of DOK7 CpG hypermethylation were obtained as 85.03%, 29.1% and 57.34% in TR, TS and normal control respectively. Significant hypermethylation were found among TR vs. TS (p < 0.001), TS vs. normal (p < 0.001) and TR vs. normal controls (p < 0.03). Online databases expression and survival analysis of DOK7 showed increasing expression in TS groups vs. TR groups which have consistency with our methylation alteration results. The sensitivity and specificity of the TR epigenetic test were determined using ROC analysis showed 89.66% and 96.77% respectively and showed that 37.5% above hypermethylation is at risk for TR and breast cancer recurrence. There is a significant difference in the methylation ratio of DOK7 between tamoxifen resistant and tamoxifen sensitive groups that may be useful in the early diagnosis of tamoxifen resistance in BC cases and cancer recurrence prevention.

Effect of Sodium Butyrate and Epigallocatechin-3-Gallate on the Genes Expression of Intrinsic Apoptotic Pathway on PA-TU-8902, CFPAC-1, and CAPAN-1 Human Pancreatic Cancer Cell Lines: Epi-drugs and Intrinsic Apoptotic Pathway in Pancreatic Cancer.

Background: Histone deacetylase inhibitors (HDACIs) are novel anticancer agents that induce cell death and cycle arrest. Several studies reported that HDACIs induce apoptosis via two well-defined intrinsic/mitochondrial and death receptor pathways. In addition to HDACIs, DNA methyltransferase inhibitors effectively revert the promoter hypermethylation of tumor suppressor genes and apoptosis induction. The current study aimed to investigate the effect of sodium butyrate and epigallocatechin-3-gallate (EGCG) on the genes expression of the intrinsic pathway (BAX, BAK, APAF1, Bcl-2, and Bcl-xL), p21, and p53 on PA-TU-8902, CFPAC-1, and CAPAN-1 human pancreatic cancer cell lines. Materials and Methods:The PA-TU-8902, CFPAC-1, and CAPAN-1 cells were treated with sodium butyrate and EGCG. To determine cell viability, cell apoptosis, and the relative gene expression level, the 3-(4,4-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry, and real-time quantitative reverse transcription polymerase chain reaction were done, respectively. Results: Both compounds changed the expression levels of the mentioned genes in a p53-dependent and -independent manner, which induced cell apoptosis and inhibited cell growth in all three cell lines. Conclusion: We indicated that sodium butyrate and EGCG could induce apoptosis in human pancreatic cancer cell lines.[GMJ.2022;11:e2248].

Emerging role of different DNA methyltransferases in the pathogenesis of cancer.

DNA methylation is one of the most essential epigenetic mechanisms to regulate gene expression. DNA methyltransferases (DNMTs) play a vital role in DNA methylation in the genome. In mammals, DNMTs act with some elements to regulate the dynamic DNA methylation patterns of embryonic and adult cells. Conversely, the aberrant function of DNMTs is frequently the hallmark in judging cancer, including total hypomethylation and partial hypermethylation of tumor suppressor genes (TSGs), which improve the malignancy of tumors, aggravate the ailment for patients, and significantly exacerbate the difficulty of cancer therapy. Since DNA methylation is reversible, currently, DNMTs are viewed as an important epigenetic target for drug development. However, the impression of DNMTs on cancers is still controversial, and therapeutic methods targeting DNMTs remain under exploration. This review mainly summarizes the relationship between the main DNMTs and cancers as well as regulatory mechanisms and clinical applications of DNMTs in cancer and highlights several forthcoming strategies for targeting DNMTs.

DNA Methylation of HOXA11 Gene as Prognostic Molecular Marker in Human Gastric Adenocarcinoma.

Hypermethylation of tumor suppressor genes and hypomethylation of oncogenes might be identified as possible biomarkers in gastric cancer (GC). We aimed to assess the DNA methylation status of selected genes in GC tissue samples and evaluate these genes’ prognostic importance on patient survival. Patients (99) diagnosed with GC and who underwent gastrectomy were included. We selected a group of genes (RAD51B, GFRA3, AKR7A3, HOXA11, TUSC3, FLI1, SEZ6L, GLDC, NDRG) which may be considered as potential tumor suppressor genes and oncogenes. Methylation of the HOXA11 gene promoter was significantly more frequent in GC tumor tissue (p = 0.006) than in healthy gastric mucosa. The probability of surviving longer (71.2 months (95% CI 57–85.3) vs. 44.3 months (95% CI 34.8–53.9)) was observed with unmethylated HOXA11 promoter in cancer tissues. Survival in patients with a methylation of HOXA11 promoter either in healthy gastric mucosa or gastric cancer tissue was twice as high as in patients with a methylation of HOXA11 promoter in both healthy gastric mucosa and cancer tissue (61.2 months (95% CI 50.9–71.4) vs. 28.5 months (95% CI 20.8–36.2)). Multivariate Cox analysis revealed the HOXA11 methylation as significantly associated with patients’ survival (HR = 2.4, 95% CI 1.19–4.86). Our results suggest that the HOXA11 gene might be a potential prognostic molecular marker in patients with gastric adenocarcinoma.

A phase 1 study of NTX-301, an oral DNMT1 inhibitor, in patients with MDS and AML (trial in progress).

TPS7077 Background: Patients with relapsed and/or refractory (r/r) myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), and acute myeloid leukemia (AML) who are not candidates for stem-cell transplantation have dismal outcomes. Novel therapies are needed. The nucleoside analogue NTX-301 (5-aza-4’-thio-2’-deoxycytidine or Aza-TdC) is incorporated into DNA, where it engages the active site of DNA methyltransferase I (DNMT1), a maintenance methyltransferase that contributes to the hypermethylation and silencing of tumor suppressor genes. DNMT1 can become trapped in a covalent complex with DNA, thus depleting free enzyme and inhibiting the normal maintenance methylation of CpG sites, resulting in re-activation of tumor suppressor genes. Preclinical data suggest a correlation between NTX-301 activity in leukemia xenograft models and decreased levels of DNMT1. NTX-301 offers an improvement over traditional DNA methyltransferase inhibitors (azacitidine, decitabine) by virtue of a higher incorporation rate into DNA at lower levels of cytotoxicity. Methods: This is a phase 1, open-label, multi-center, dose-escalation study to assess the safety, tolerability, and recommended myeloid monotherapy dose of NTX-301 in patients with r/r myeloid neoplasms. Patients ≥ 18 years with r/r higher-risk MDS (intermediate, high, very-high risk by IPSS-R), high-risk CMML (int-2/high by CPSS or CPSS-mol), or AML (marrow blasts ≤ 30% or WBC &lt; 20,000 cells/µL without leukoreduction) are eligible. Other key eligibility criteria: Eastern Cooperative Oncology Group performance status 0–2; adequate cardiac, renal, and liver function; and resolved acute effects of any prior therapy. Successive cohorts of patients will receive escalating doses of NTX-301 starting from 2 mg QD. Each cycle is 21 days in duration (treatment for 5 days/week x 2 weeks and 1 week off). The trial incorporates accelerated titration design for dose level 1 and 2 followed by traditional 3+3 dose escalation design (3-6 patients per cohort) from dose level 3. Intrapatient dose escalation is allowed. NTX-301 treatment will continue until disease progression or unacceptable toxicity, whichever occurs first. Primary endpoints: incidence and severity of adverse events, dose-limiting toxicities (DLTs), maximum tolerated dose (MTD), biologically effective dose (BED). Secondary endpoints: PK parameters of NTX-301, PD assessment through global hypomethylation assay and other markers in blood and marrow, and clinical efficacy [overall response rate (ORR), complete remission (CR), marrow CR (mCR), partial remission (PR), stable disease (SD), hematologic improvement (HI) per mIWG criteria]. After the MTD/BED is identified, the safety and efficacy of NTX-301 will be explored further in selected myeloid malignancies. The study began enrolling patients in January 2021 and is still recruiting. Clinical trial information: NCT04167917.

DNA hypermethylation modification promotes the development of hepatocellular carcinoma by depressing the tumor suppressor gene ZNF334

DNA methylation plays a pivotal role in the development and progression of tumors. However, studies focused on the dynamic changes of DNA methylation in the development of hepatocellular carcinoma (HCC) are rare. To systematically illustrate the dynamic DNA methylation alternation from premalignant to early-stage liver cancer with the same genetic background, this study enrolled 5 HBV-related patients preceded with liver cirrhosis, pathologically identified as early-stage HCC with dysplastic nodules. Liver fibrosis tissues, dysplastic nodules and early HCC tissues from these patients were used to measure DNA methylation. Here, we report significant differences in the DNA methylation spectrum among the three types of tissues. In the early stage of HCC, DNA hypermethylation of tumor suppressor genes is predominant. Additionally, DNA hypermethylation in the early stage of HCC changes the binding ability of transcription factor TP53 to the promoter of tumor suppressor gene ZNF334, and inhibits the expression of ZNF334 at the transcription level. Furthermore, through a series of in vivo and in vitro experiments, we have clarified the exacerbation effect of tumor suppressor gene ZNF334 deletion in the occurrence of HCC. Combined with clinical data, we found that the overall survival and relapse-free survival of patients with high ZNF334 expression are significantly longer. Thus, we partly elucidated a sequential alternation of DNA methylation modification during the occurrence of HCC, and clarified the biological function and regulatory mechanism of the tumor suppressor gene ZNF334, which is regulated by related DNA methylation sites. Our study provides a new target and clinical evidence for the early diagnosis and sheds light on the precise treatment of liver cancer.