Silencing Of Tumor Suppressor Genes Research Articles

UHRF1 poly-auto-ubiquitination induced by the anti-cancer drug, thymoquinone, is involved in the DNA repair machinery recruitment.

DNA methylation is one of the most important epigenetic mark involved in many physiologic cellular processes and pathologies. During mitosis, the transmission of DNA methylation patterns from a mother to the daughter cells is ensured through the action of the Ubiquitin-like, containing PHD and RING domains, 1/DNA methyltransferase 1 (UHRF1/DNMT1) tandem. UHRF1 is involved in the silencing of many tumor suppressor genes (TSGs) via mechanisms that remain largely to be deciphered. The present study investigated the role and the regulation of UHRF1 poly-ubiquitination induced by thymoquinone, a natural anti-cancer drug, known to enhance or re-activate the expression of TSGs. We found that the auto-ubiquitination of UHRF1, induced by TQ, is mediated by reactive oxygen species, and occurs following DNA damage. We demonstrated that the poly-ubiquitinated form of UHRF1 is K63-linked and can still silence the tumor suppressor gene p16INK4A/CDKN2A. We further showed that TQ-induced auto-ubiquitination is mediated via the activity of Tip60. Since this latter is known as a nuclear receptor co-factor, we investigated if the glucocorticoid receptor (GR) might be involved in the regulation of UHRF1 ubiquitination. Activation of the GR, with dexamethasone, did not influence auto-ubiquitination of UHRF1. However, we could observe that TQ induced a K48-linked poly-ubiquitination of GR, probably involved in the proteosomal degradation pathway. Mass-spectrometry analysis of FLAG-HA-tagged UHRF1 identified UHRF1 partners involved in DNA repair and showed that TQ increased their association with UHRF1, suggesting that poly-ubiquitination of UHRF1 is involved in the DNA repair process. We propose that poly-ubiquitination of UHRF1 serves as a scaffold to recruit the DNA repair machinery at DNA damage sites.

Abstract 212: Repression of NKX2-1 promote epithelial-mesenchymal transition and tumor neutrophil recruitment in lung cancer progression

Abstract Background: Malignant transformation is associated with tumor aggressiveness and poor patient outcome. This cellular alteration is due to either activation of oncogenes or the silencing of tumor suppressor genes. NKX2-1 was identified as a lineage-specific gene downregulated in poorly differentiated lung adenocarcinoma (LUAD). However, the molecular mechanism and functional role of infiltrated immune cells in NKX2-1-negative tumors are not clearly understood. Methods: Clinical relevance of NKX2-1 expression and disease status were evaluated by immunohistochemical analysis of LUAD tissue arrays and the overall survival analysis of TCGA databases. In vitro and in vivo experiments were applied to confirm the role and mechanisms of NKX2-1 actions in transformed cells. The secretion of chemokines was evaluated by using chemokine array. Flow cytometry and single-cell RNA sequencing analysis of mouse tumor tissue were used to demonstrate the infiltration of neutrophils in mouse models. Results: Low expression of NKX2-1 was observed in high-grade LUAD tumors. LUAD patients who express a pattern of NKX2-1high/TGF-βlow had better survival outcomes than those with NKX2-1low/TGF-βhigh. Mechanistically, TGF-β induced the downregulation of NKX2-1 which led to enhanced epithelial-mesenchymal transition (EMT), cell motility, and CXC chemokine expression. The high release of these chemoattractants promoted the recruitment of neutrophils. Meanwhile, NKX2-1-dependent suppression of CXC chemokines gene expression may be via repressive histone methylation at the promoter region of CXC chemokines in lung cancer cells. In the in vivo syngeneic LUAD models, exacerbated neutrophil infiltration was noted. Single-cell RNA sequencing and flow cytometry both revealed that neutrophils were recruited via CXCLs/CXCR2 activation in LUAD tumors with NKX2-1 knockdown. Conclusion: Our data showed that NKX2-1 represses tumor growth, metastasis, and neutrophil recruitment via the CXCLs/CXCR2-dependent mechanisms. Notably, targeting CXCR2 in NKX2-1-negative tumors may improve LUAD patient outcomes. Citation Format: Shih-Hwa Chiou, Ping-Hsing Tsai, Mong-Lien Wang. Repression of NKX2-1 promote epithelial-mesenchymal transition and tumor neutrophil recruitment in lung cancer progression [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 212.

Abstract 5976: R9: A novel NuRD complex inhibitor for targeted therapy in triple negative breast cancer (TNBC)

Abstract Triple Negative Breast Cancer (TNBC) is a highly aggressive breast cancer subtype, disproportionately affecting young and African American populations. TNBC constitutes up to 15% of breast cancers that lack effective targeted therapy. The Nucleosome Remodeling and Deacetylase (NuRD) complex, a pivotal chromatin remodeling entity, plays a crucial role in cancer development and progression. Specifically, the NuRD complex associates with SALL4 in cancer cells, contributing to the silencing of tumor-suppressor genes like PTEN. In this study, we discover R9, a novel small-molecule nucleotide analog, a first-in-class NuRD inhibitor. Utilizing computational and cellular methods, R9 demonstrated selective engagement with RBBP4, a key NuRD complex subunit. Cellular target engagement technology has been utilized to verify direct interaction of R9 with NuRD complex as well as to guide medicinal chemistry efforts. Mass spectrometry thermal proteome profiling (TPP) affirmed RBBP4 as the primary target of R9 with minimal off-target effects. In vitro studies reveal R9 displaying robust toxicity in TNBC cells, while sparing normal tissue derived cells. Encouragingly, R9 effectively impeded TNBC tumors growth in mice without inducing toxicity. Mechanistically, we demonstrate R9 induces DNA damage in sensitive TNBC cells, and the sensitivity correlates to DNA damage-vulnerable cancer cells, indicating DNA damage is a major effector of NuRD complex inhibition in TNBCs. We further demonstrate that R9 induces DNA damage through a c-MYC dependent manner. Ongoing studies aim to elucidate key transcription factors involved in NuRD complex inhibition and the role of MYC and DNA damage in response to NuRD complex inhibition. This research positions R9 as a promising candidate for targeted therapy in TNBC, offering potential insights into the intricate molecular pathways involved in TNBC tumor progression. Citation Format: Kun Zhang, Sheeba Jacob, Mikhail Dozmorov, Ivan Babic, Elmar Nurmemmedov, Anthony C. Faber. R9: A novel NuRD complex inhibitor for targeted therapy in triple negative breast cancer (TNBC) [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 5976.

Gastric Cancer in the Era of Epigenetics.

Gastric cancer (GC) remains a significant contributor to cancer-related mortality. Novel high-throughput techniques have enlightened the epigenetic mechanisms governing gene-expression regulation. Epigenetic characteristics contribute to molecular taxonomy and give rise to cancer-specific epigenetic patterns. Helicobacter pylori (Hp) infection has an impact on aberrant DNA methylation either through its pathogenic CagA protein or by inducing chronic inflammation. The hypomethylation of specific repetitive elements generates an epigenetic field effect early in tumorigenesis. Epstein-Barr virus (EBV) infection triggers DNA methylation by dysregulating DNA methyltransferases (DNMT) enzyme activity, while persistent Hp-EBV co-infection leads to aggressive tumor behavior. Distinct histone modifications are also responsible for oncogene upregulation and tumor-suppressor gene silencing in gastric carcinomas. While histone methylation and acetylation processes have been extensively studied, other less prevalent alterations contribute to the development and migration of gastric cancer via a complex network of interactions. Enzymes, such as Nicotinamide N-methyltransferase (NNMT), which is involved in tumor's metabolic reprogramming, interact with methyltransferases and modify gene expression. Non-coding RNA molecules, including long non-coding RNAs, circular RNAs, and miRNAs serve as epigenetic regulators contributing to GC development, metastasis, poor outcomes and therapy resistance. Serum RNA molecules hold the potential to serve as non-invasive biomarkers for diagnostic, prognostic or therapeutic applications. Gastric fluids represent a valuable source to identify potential biomarkers with diagnostic use in terms of liquid biopsy. Ongoing clinical trials are currently evaluating the efficacy of next-generation epigenetic drugs, displaying promising outcomes. Various approaches including multiple miRNA inhibitors or targeted nanoparticles carrying epigenetic drugs are being designed to enhance existing treatment efficacy and overcome treatment resistance.

Abstract PR-008: Modeling epigenetic lesions that cause gliomas

Abstract PR-008: Modeling epigenetic lesions that cause gliomas

Preclinical Evaluation of NTX-301, a Novel DNA Hypomethylating Agent in Ovarian Cancer.

DNA methylation causes silencing of tumor-suppressor and differentiation-associated genes, being linked to chemoresistance. Previous studies demonstrated that hypomethylating agents (HMA) resensitize ovarian cancer to chemotherapy. NTX-301 is a highly potent and orally bioavailable HMA, in early clinical development. The antitumor effects of NTX-301 were studied in ovarian cancer models by using cell viability, stemness and ferroptosis assays, RNA sequencing, lipidomic analyses, and stimulated Raman spectroscopy. Ovarian cancer cells (SKOV3, IC50 = 5.08 nmol/L; OVCAR5 IC50 = 3.66 nmol/L) were highly sensitive to NTX-301 compared with fallopian tube epithelial cells. NTX-301 downregulated expression of DNA methyltransferases 1-3 and induced transcriptomic reprogramming with 15,000 differentially expressed genes (DEG, P < 0.05). Among them, Gene Ontology enrichment analysis identified regulation of fatty acid biosynthesis and molecular functions related to aldehyde dehydrogenase (ALDH) and oxidoreductase, known features of cancer stem cells. Low-dose NTX-301 reduced the ALDH(+) cell population and expression of stemness-associated transcription factors. Stearoyl-coenzyme A desaturase 1 (SCD), which regulates production of unsaturated fatty acids (UFA), was among the top DEG downregulated by NTX-301. NTX-301 treatment decreased levels of UFA and increased oxidized lipids, and this was blunted by deferoxamine, indicating cell death via ferroptosis. NTX-301-induced ferroptosis was rescued by oleic acid. In vivo, monotherapy with NTX-301 significantly inhibited ovarian cancer and patient-derived xenograft growth (P < 0.05). Decreased SCD levels and increased oxidized lipids were detected in NTX-301-treated xenografts. NTX-301 is active in ovarian cancer models. Our findings point to a new mechanism by which epigenetic blockade disrupts lipid homeostasis and promotes cancer cell death.

Recent insights into the functions and mechanisms of antisense RNA: emerging applications in cancer therapy and precision medicine.

The antisense RNA molecule is a unique DNA transcript consisting of 19-23 nucleotides, characterized by its complementary nature to mRNA. These antisense RNAs play a crucial role in regulating gene expression at various stages, including replication, transcription, and translation. Additionally, artificial antisense RNAs have demonstrated their ability to effectively modulate gene expression in host cells. Consequently, there has been a substantial increase in research dedicated to investigating the roles of antisense RNAs. These molecules have been found to be influential in various cellular processes, such as X-chromosome inactivation and imprinted silencing in healthy cells. However, it is important to recognize that in cancer cells; aberrantly expressed antisense RNAs can trigger the epigenetic silencing of tumor suppressor genes. Moreover, the presence of deletion-induced aberrant antisense RNAs can lead to the development of diseases through epigenetic silencing. One area of drug development worth mentioning is antisense oligonucleotides (ASOs), and a prime example of an oncogenic trans-acting long noncoding RNA (lncRNA) is HOTAIR (HOX transcript antisense RNA). NATs (noncoding antisense transcripts) are dysregulated in many cancers, and researchers are just beginning to unravel their roles as crucial regulators of cancer's hallmarks, as well as their potential for cancer therapy. In this review, we summarize the emerging roles and mechanisms of antisense RNA and explore their application in cancer therapy.

Methyl-CpG Binding Domain Protein MBD2 Is a Targetable Vulnerability in TET2 Mutant Myeloid Neoplasia

Background &amp; Significance. Epigenetic dysregulation leading to acquired disorganization of CpG methylation (mCpG) is a hallmark of cancer in general and myeloid neoplasia (MN) in particular. TET dioxygenases (TET1, TET2 and TET3) progressively oxidize 5-methyl cytosine (mC) to facilitate CpG demethylation, a process critical to efficient transcription, lineage differentiation, and proliferation. Loss of function TET2 mutation ( TET2 MT), the predominant TET in HSPCs, isone of the most common genetic lesions in MN and often occurs early in MDS/AML as a founder lesion. It is frequently observed in clonal hematopoiesis of indeterminate potential (CHIP) 1,2, a prodromal condition linked with increased risk of MN, coronary heart disease 3, and pulmonary arterial hypertension 4. TET2 MT results in abnormal accumulation of mC in the genome resulting in loss of lineage plasticity due to transcriptional repression of differentiation-promoting genes and tumor suppressor genes (TSGs), contributing to a volatile pre-neoplastic state. Indeed, the accumulation of mC at otherwise unmethylated promoter- and enhancer-associated CpGs is a hallmark cancer 2,5. The epigenetic reader mCpG binding domain protein 2 (MBD2) specifically recognizes mCpG, thereby modulating regional gene transcription 6. This modulation occurs through the recruitment of co-repressor complexes which deliver enzymatic machinery to reorganize chromatin into compact, inaccessible, and stably silenced regions. This tightly regulated process, required for normal development and cell lineage differentiation, is hijacked by malignant cells to maintain a pro-oncogenic transcriptional program. Therefore, MBD2 inhibition may restore the normal hematopoiesis in TET2 MT HSPCs and may represent a selective therapeutic target. Methods. Genetic and epigenetic in vitro and in vivo model systems of clonal evolution including competitive repopulation assays and CRISPR-engineered cells coupled with NGS and biochemical analysis were used to establish the functional relevance of MBD2 in the clonal evolution of TET2 MT HSPCs. In addition, gene knockout or knockdown (ko/kd) were used to establish MBD2 as a novel therapeutic vulnerability in TET2 MT MN. Results. Here we report that loss of TET activity, either by TET2 MT or by neomorphic IDH1/2 MT, selectively enriches promoter/enhancer mCpG contributing to malignant evolution of TET2 MT HSPCs that creates sensitivity to MBD2 inhibition. TET2 MT creates regional accumulation of mC that recruits the MBD2-NuRd complex, resulting in silencing of TSGs as demonstrated via MBD2 chromatin immunoprecipitation assay. MBD2 ko/kd in TET2 MT cells (SIG-M5, OCIAML-5) or isogenic CRISPR-engineered leukemic cells (THP1, K562) resulted in the termination of the malignant program as reflected in a complete loss of colony forming ability in vitro. Orthotropic transplant of SIG-M5 cells (10 5 cells/mice) created leukemia in SGM3-NSGS mice with a maximum survival of 45 days, however, MBD2 ko in transplanted cells resulted in 100% disease-free survival of these mice. To further test if Mbd2 loss is sufficient to restore normal hematopoiesis and if it can abrogate the proliferative advantage of Tet2 -/- HSPCs, we developed a Tet2 -/-;Mbd2 -/- double ko (DKO) murine model by crossing Tet2 -/-with Mbd2 -/- mice. The DKO mice developed normally with restored hematopoiesis within the myeloid lineage compared to Tet2 -/-. Most importantly, the proliferative advantage of Tet2 -/-HSPCs was ablated, as reflected in the mutant fraction of total WBC and the restoration of CD11b + myeloid cells following Mbd2 loss in competitive bone marrow repopulation assays ( Fig. A).This restoration was mainly driven by the transcriptional rescue of differentiation-promoting genes which were silenced by promoter methylation in Tet2 -/- HSPCs ( Fig. B). Discussion &amp; Conclusion. Here, for the first time, we demonstrate that MBD2 is required for maintaining the undifferentiated state of TET2 MT HSPCs with myeloid skewing. We provide several lines of evidence in human cells as well as in murine models that MBD2 is a targetable vulnerability in TET2 MT-associated leukemia. Our current findings provide a rationale for development of novel and potent non-cytotoxic therapeutic agents targeting MBD2 and their application in a biomarker-driven fashion for a large proportion of leukemias.

Genome-Wide Methylation Sensitive Elements Demarcate Targetable Regulatory Elements for Controllable Gene Activation

There are multiple published studies correlating DNA methylation with gene expression, but to date, most are associative in nature without precise localization of causal elements. Multiple CpG destinations have been described with inconsistent evidence supporting their causal association with transcription. This has hampered adoption and use of methylation signatures as biomarkers or for targeted therapies. This has had consequent effects in hindering mechanistic understanding in how global demethylation agents lead to responses in some patients, but not others. With this state of the literature in mind, we set out to better understand and locate causal methylation regulatory loci genome-wide. We hypothesized that causal methylation regulatory elements possess heightened sensitivity to regulatory signals such as “demethylate”; signals that can be introduced using hypo-methylating agents (HMAs) typically used in treatment of Myelodysplastic syndromes and Acute Myeloid Leukemia. We thus analyzed 19 whole-genomic bisulfite sequenced sample pairs (pre/post-demethylation in patients, cell lines, and in murine in vivo samples) and identified previously undescribed DNA methylation sensitive elements throughout the genome, which we call Methylation Mesas (MMs) based on the profile shape observed in naïve and post-HMA treated sample. These narrow-width MMs range typically between 45-300bp wide and are present throughout the genome with greater abundance in noncoding and intragenic regions than in promoter-CpG islands. We also demonstrate that MM show an up to 90% concordant overlap with primed and active histone marks by means of a compiled histone mark footprint map using 60 human and 59 murine ENCODE datasets for each respective species, suggesting a functional role for MMs as regulatory elements with transcriptional potential. While HMA's enable identification of MM's genome-wide, their mode of action renders them unsuitable for ascertaining the direct transcriptional potential of any singular locus. Therefore, in order to ascertain the causal role of MM for transcriptional regulation, we developed a fine-resolution targeted demethylation technology(CRISPR-DiR), leveraging our previously published findings that DNA methyltransferase I (DNMT1) can be blocked by DNMT1-interacting RNAs (DiR). Compared with other targeted demethylation technologies with wider effect windows (eg. CRISPR-TET1), CRISPR-DiR shows superior locus specificity thus serving as an ideal tool to identify causal regulatory elements. Using tumor suppressor p16 as an example, we elucidated that demethylation of a single MM in the first exon is sufficient to trigger locus and distal chromatin rewiring events that natively initiate expression to a significantly greater extent than promoter CpG island targeting alone. Similar CRISPR-DiR activation is also observed in four additional, natively silenced tumor suppressor genes, one of which has no annotated CpG island. In summary, we have identified a previously undescribed narrow-width DNA methylation sensitive regulatory element, consistent across all epigenomes investigated, located largely independent of the promoter CpG island dogma, yet initiating gene transcription more potently. Identification of said elements paves the way for future studies to better understand the mechanisms of how mutations in DNA methyltransferases lead to disease, how global hypomethylating agents can lead to a response in diseases such as MDS and AML but can also activate oncogenes, and finally development of targeted therapies.

Epigenetic Regulation in Oral Squamous Cell Carcinoma Microenvironment: A Comprehensive Review

Oral squamous cell carcinoma (OSCC) is a prevalent and significant type of oral cancer that has far-reaching health implications worldwide. Epigenetics, a field focused on studying heritable changes in gene expression without modifying DNA sequence, plays a pivotal role in OSCC. Epigenetic changes, encompassing DNA methylation, histone modifications, and miRNAs, exert control over gene activity and cellular characteristics. In OSCC, aberrant DNA methylation of tumor suppressor genes (TSG) leads to their inactivation, subsequently facilitating tumor growth. As a result, distinct patterns of gene methylation hold promise as valuable biomarkers for the detection of OSCC. Oral cancer treatment typically involves surgery, radiation therapy, and chemotherapy, but even with these treatments, cancer cells cannot be effectively targeted and destroyed. Researchers are therefore exploring new methods to target and eliminate cancer cells. One promising approach is the use of epigenetic modifiers, such as DNA methyltransferase (DNMT) inhibitors and histone deacetylase (HDAC) inhibitors, which have been shown to modify abnormal epigenetic patterns in OSCC cells, leading to the reactivation of TSGs and the suppression of oncogenes. As a result, epigenetic-targeted therapies have the potential to directly alter gene expression and minimize side effects. Several studies have explored the efficacy of such therapies in the treatment of OSCC. Although studies have investigated the efficacy of epigenetic therapies, challenges in identifying reliable biomarkers and developing effective combination treatments are acknowledged. Of note, epigenetic mechanisms play a significant role in drug resistance in OSCC and other cancers. Aberrant DNA methylation can silence tumor suppressor genes, while alterations in histone modifications and chromatin remodeling affect gene expression related to drug metabolism and cell survival. Thus, understanding and targeting these epigenetic processes offer potential strategies to overcome drug resistance and improve the efficacy of cancer treatments in OSCC. This comprehensive review focuses on the complex interplay between epigenetic alterations and OSCC cells. This will involve a deep dive into the mechanisms underlying epigenetic modifications and their impact on OSCC, including its initiation, progression, and metastasis. Furthermore, this review will present the role of epigenetics in the treatment and diagnosis of OSCC.

Compound AC1Q3QWB upregulates CDKN1A and SOX17 by interrupting the HOTAIR—EZH2 interaction and enhances the efficacy of tazemetostat in endometrial cancer

Endometrial cancer (EC) is a common malignancy of the female reproductive system, with an escalating incidence. Recurrent/metastatic EC presents a poor prognosis. The interaction between the long non-coding RNA (lncRNA) HOTAIR and the polycomb repressive complex 2 (PRC2) induces abnormal silencing of tumor suppressor genes, exerting a pivotal role in tumorigenesis. We have previously discovered AC1Q3QWB (AQB), a small-molecule compound targeting HOTAIR-EZH2 interaction. In the present study, we unveil that AQB selectively hampers the interaction between HOTAIR and EZH2 within EC cells, thus reversing the epigenetic suppression of tumor suppressor genes. Furthermore, our findings demonstrate AQB's synergistic effect with tazemetostat (TAZ), an EZH2 inhibitor, significantly boosting the expression of CDKN1A and SOX17. This, in turn, induces cell cycle arrest and impedes EC cell proliferation, migration, and invasion. In vivo experiments further validate AQB's potential by enhancing TAZ's anti-tumor efficacy at lower doses. Our results advocate AQB, a recently discovered small-molecule inhibitor, as a promising agent against EC cells. When combined with TAZ, it offers a novel therapeutic strategy for EC treatment.

Natural and Synthetic Anticancer Epidrugs Targeting the Epigenetic Integrator UHRF1.

Cancer is one of the leading causes of death worldwide, and its incidence and mortality are increasing each year. Improved therapeutic strategies against cancer have progressed, but remain insufficient to invert this trend. Along with several other risk factors, abnormal genetic and epigenetic regulations play a critical role in the initiation of cellular transformation, as well as tumorigenesis. The epigenetic regulator UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) is a multidomain protein with oncogenic abilities overexpressed in most cancers. Through the coordination of its multiple domains and other epigenetic key players, UHRF1 regulates DNA methylation and histone modifications. This well-coordinated dialogue leads to the silencing of tumor-suppressor genes (TSGs) and facilitates tumor cells' resistance toward anticancer drugs, ultimately promoting apoptosis escape and uncontrolled proliferation. Several studies have shown that the downregulation of UHRF1 with natural compounds in tumor cells induces the reactivation of various TSGs, inhibits cell growth, and promotes apoptosis. In this review, we discuss the underlying mechanisms and the potential of various natural and synthetic compounds that can inhibit/minimize UHRF1's oncogenic activities and/or its expression.

PVT1 interacts with polycomb repressive complex 2 to suppress genomic regions with pro-apoptotic and tumour suppressor functions in multiple myeloma.

Multiple myeloma is a heterogeneous hematological disease that originates from the bone marrow and is characterized by the monoclonal expansion of malignant plasma cells. Despite novel therapies, multiple myeloma remains clinically challenging. A common feature among patients with poor prognosis is the increased activity of the epigenetic silencer EZH2, which is the catalytic subunit of the PRC2. Interestingly, the recruitment of PRC2 lacks sequence specificity and, to date, the molecular mechanisms that define which genomic locations are destined for PRC2-mediated silencing remain unknown. The presence of a long non-coding RNA (lncRNA)-binding pocket on EZH2 suggests that lncRNA could potentially mediate PRC2 recruitment to specific genomic regions. Here, we coupled RNA immunoprecipitation sequencing, RNA-sequencing and chromatin immunoprecipitation-sequencing analysis of human multiple myeloma primary cells and cell lines to identify potential lncRNA partners to EZH2. We found that the lncRNA plasmacytoma variant translocation 1 (PVT1) directly interacts with EZH2 and is overexpressed in patients with a poor prognosis. Moreover, genes predicted to be targets of PVT1 exhibited H3K27me3 enrichment and were associated with pro-apoptotic and tumor suppressor functions. In fact, PVT1 inhibition independently promotes the expression of the PRC2 target genes ZBTB7C, RNF144A and CCDC136. Altogether, our work suggests that PVT1 is an interacting partner in PRC2-mediated silencing of tumor suppressor and pro-apoptotic genes in multiple myeloma, making it a highly interesting potential therapeutic target.

Thymoquinone Enhances Apoptosis of K562 Chronic Myeloid Leukemia Cells through Hypomethylation of SHP-1 and Inhibition of JAK/STAT Signaling Pathway.

The epigenetic silencing of tumor suppressor genes (TSGs) is critical in the development of chronic myeloid leukemia (CML). SHP-1 functions as a TSG and negatively regulates JAK/STAT signaling. Enhancement of SHP-1 expression by demethylation provides molecular targets for the treatment of various cancers. Thymoquinone (TQ), a constituent of Nigella sativa seeds, has shown anti-cancer activities in various cancers. However, TQs effect on methylation is not fully clear. Therefore, the aim of this study is to assess TQs ability to enhance the expression of SHP-1 through modifying DNA methylation in K562 CML cells. The activities of TQ on cell cycle progression and apoptosis were evaluated using a fluorometric-red cell cycle assay and Annexin V-FITC/PI, respectively. The methylation status of SHP-1 was studied by pyrosequencing analysis. The expression of SHP-1, TET2, WT1, DNMT1, DNMT3A, and DNMT3B was determined using RT-qPCR. The protein phosphorylation of STAT3, STAT5, and JAK2 was assessed using Jess Western analysis. TQ significantly downregulated the DNMT1 gene, DNMT3A gene, and DNMT3B gene and upregulated the WT1 gene and TET2 gene. This led to hypomethylation and restoration of SHP-1 expression, resulting in inhibition of JAK/STAT signaling, induction of apoptosis, and cell cycle arrest. The observed findings imply that TQ promotes apoptosis and cell cycle arrest in CML cells by inhibiting JAK/STAT signaling via restoration of the expression of JAK/STAT-negative regulator genes.

Crosstalk between miRNAs and DNA Methylation in Cancer

miRNAs are some of the most well-characterized regulators of gene expression. Integral to several physiological processes, their aberrant expression often drives the pathogenesis of both benign and malignant diseases. Similarly, DNA methylation represents an epigenetic modification influencing transcription and playing a critical role in silencing numerous genes. The silencing of tumor suppressor genes through DNA methylation has been reported in many types of cancer and is associated with tumor development and progression. A growing body of literature has described the crosstalk between DNA methylation and miRNAs as an additional layer in the regulation of gene expression. Methylation in miRNA promoter regions inhibits its transcription, while miRNAs can target transcripts and subsequently regulate the proteins responsible for DNA methylation. Such relationships between miRNA and DNA methylation serve an important regulatory role in several tumor types and highlight a novel avenue for potential therapeutic targets. In this review, we discuss the crosstalk between DNA methylation and miRNA expression in the pathogenesis of cancer and describe how miRNAs influence DNA methylation and, conversely, how methylation impacts the expression of miRNAs. Finally, we address how these epigenetic modifications may be leveraged as biomarkers in cancer.

Ginseng mediates its anticancer activity by inhibiting the expression of DNMTs and reactivating methylation-silenced genes in colorectal cancer.

Developing safe and effective therapeutic modalities remains a critical challenge for improving the prognosis of patients with colorectal cancer (CRC). In this regard, targeting epigenetic regulation in cancers has recently emerged as a promising therapeutic approach. Since several natural compounds have recently been shown to be important epigenetic modulators, we hypothesized that Ginseng might exert its anticancer activity by regulating DNA methylation alterations in CRC. In this study, a series of cell culture studies were conducted, followed by their interrogation in patient-derived 3D organoid models to evaluate Ginseng's anticancer activity in CRC. Genome-wide methylation alterations were interrogated by undertaking MethylationEpic BeadChip microarrays. First, 50% inhibitory concentrations (IC50) were determined by cell viability assays, and subsequent Ginseng treatment demonstrated a significant anticancer effect on clonogenicity and cellular migration in CRC cells. Treatment with Ginseng potentiated cellular apoptosis through regulation of apoptosis-related genes in CRC cells. Furthermore, Ginseng treatment downregulated the expression of DNA methyltransferases (DNMTs) and decreased the global DNA methylation levels in CRC cells. The genome-wide methylation profiling identified Ginseng-induced hypomethylation of transcriptionally silenced tumor suppressor genes. Finally, cell culture-based findings were successfully validated in patient-derived 3D organoids. In conclusion, we demonstrate that Ginseng exerts its antitumorigenic potential by regulating cellular apoptosis via the downregulation of DNMTs and reversing the methylation status of transcriptionally silenced genes in CRC.

Epigenetic reactivation of tumor suppressor genes with CRISPRa technologies as precision therapy for hepatocellular carcinoma

BackgroundEpigenetic silencing of tumor suppressor genes (TSGs) is a key feature of oncogenesis in hepatocellular carcinoma (HCC). Liver-targeted delivery of CRISPR-activation (CRISPRa) systems makes it possible to exploit chromatin plasticity, by reprogramming transcriptional dysregulation.ResultsUsing The Cancer Genome Atlas HCC data, we identify 12 putative TSGs with negative associations between promoter DNA methylation and transcript abundance, with limited genetic alterations. All HCC samples harbor at least one silenced TSG, suggesting that combining a specific panel of genomic targets could maximize efficacy, and potentially improve outcomes as a personalized treatment strategy for HCC patients. Unlike epigenetic modifying drugs lacking locus selectivity, CRISPRa systems enable potent and precise reactivation of at least 4 TSGs tailored to representative HCC lines. Concerted reactivation of HHIP, MT1M, PZP, and TTC36 in Hep3B cells inhibits multiple facets of HCC pathogenesis, such as cell viability, proliferation, and migration.ConclusionsBy combining multiple effector domains, we demonstrate the utility of a CRISPRa toolbox of epigenetic effectors and gRNAs for patient-specific treatment of aggressive HCC.

Genomic Loss and Epigenetic Silencing of the FOSL1 Tumor Suppressor Gene in Radiation-induced Neoplastic Transformation of Human CGL1 Cells Alters the Tumorigenic Phenotype In Vitro and In Vivo.

The CGL1 human hybrid cell system has been utilized for many decades as an excellent cellular tool for investigating neoplastic transformation. Substantial work has been done previously implicating genetic factors related to chromosome 11 to the alteration of tumorigenic phenotype in CGL1 cells. This includes candidate tumor suppressor gene FOSL1, a member of the AP-1 transcription factor complex which encodes for protein FRA1. Here we present novel evidence supporting the role of FOSL1 in the suppression of tumorigenicity in segregants of the CGL1 system. Gamma-induced mutant (GIM) and control (CON) cells were isolated from 7 Gy gamma-irradiated CGL1s. Western, Southern and Northern blot analysis were utilized to assess FOSL1/FRA1 expression as well as methylation studies. GIMs were transfected to re-express FRA1 and in vivo tumorigenicity studies were conducted. Global transcriptomic microarray and RT-qPCR analysis were used to further characterize these unique cell segregants. GIMs were found to be tumorigenic in vivo when injected into nude mice whereas CON cells were not. GIMs show loss of Fosl/FRA1 expression as confirmed by Western blot. Southern and Northern blot analysis further reveals that FRA1 reduction in tumorigenic CGL1 segregants is likely due to transcriptional suppression. Results suggest that radiation-induced neoplastic transformation of CGL1 is in part due to silencing of the FOSL1 tumor suppressor gene promoter by methylation. The radiation-induced tumorigenic GIMs transfected to re-express FRA1 resulted in suppression of subcutaneous tumor growth in nude mice in vivo. Global microarray analysis and RT-qPCR validation elucidated several hundred differentially expressed genes. Downstream analysis reveals a significant number of altered pathways and enriched Gene Ontology terms genes related to cellular adhesion, proliferation, and migration. Together these findings provide strong evidence that FRA1 is a tumor suppressor gene deleted and epigenetically silenced after ionizing radiation-induced neoplastic transformation in the CGL1 human hybrid cell system.

Abstract 6023: Plasma methylome landscape across the spectrum of prostate cancer progression, and differentially methylated region (DMR) based biomarkers of hormonal therapy failure in metastatic hormone-sensitive prostate cancer

Abstract Background: DNA silencing of gene expression can affect tumorigenesis, treatment outcomes and prognosis. Epigenetic silencing of tumor suppressor genes in prostate cancer detected in plasma at different stages of progression and its effect on clinical outcomes has not been determined. In a cohort study across different stages of prostate cancer (PC) progression, we performed plasma cell-free DNA (cfDNA)-based epigenomic profiling for determining the landscape of methylation and Differentially Methylated Region (DMR) classifiers and the potential impact on clinical outcomes in metastatic hormone-sensitive prostate cancer (mHSPC) state. Methods: Plasma was collected prospectively in a longitudinal cohort study of 108 PC patients (pts) of which 24 had non-metastatic Local PC; 28 had mHSPC; 56 had mCRPC. cfDNA was used for enzymatic methylation sequencing (EM-seq) of a targeted panel of genes (N=441) implicated in prostate cancer biology. Methylation status at CpG islands of these genes were examined using Twist targeted cfDNA methylation assay. The Bismark program was used for methylation calls. Temporal trends of differentially methylated regions (DMRs) were identified in different states of progression by comparing the methylation status of target genes in localized PC, mHSPC and mCRPC states using a False Discovery Rate (FDR-q-value) of 0.2 or less. DMRs uniquely associated with mHSPC state were identified and determination of genes with DMRs in pts experiencing early failure of androgen deprivation therapy (ADT)-based therapies was performed. Early failure was defined as progression on ADT within 12 months. Results: We identified 304/441 genes that were differentially methylated between localized PC and advanced PC (mHSPC and mCRPC) (FDR&amp;lt;0.2), of which 237 genes differed between mHSPC and mCRPC (p&amp;lt;0.05) states. mHSPC pts were serially profiles before and after 3 months of ADT-based therapies. All 28 pts experienced a PSA response at 3-months and 112/237 genes showed significant change in methylation status in these serial samples taken before/after 3 months of ADT-based therapies. The median follow up of the cohort was 18 months (range 10-24). 6/28 mHSPC pts experienced early ADT failure (&amp;lt;12 months; median 10 months). 8/112 genes were significantly associated with early failures and included DMRs in RB1, FOXA1, SOX11. GSC, ALDH1L1, SCGB3A1, FLT4, and PTPRN2. Conclusions. A targeted cfDNA plasma liquid-biopsy based epigenomic profiling of PC cancer related genes was able to capture significant differences in DMRs across different states of cancer progression. Candidate differentially expressed CpG methylome gene biomarkers detected in plasma also identified biomarkers associated with early ADT-based treatment failure in mHSPC. Citation Format: Manish Kohli, Jodie Wong, Yijun Tian, Amir Bitaraf, Claire Hanson, Matt Larsen, Jennifer Lloyd, Julia Batten, Neeraj Agarwal, Umang Swami, Benjamin Maughan, Sumati Gupta, Jonathan Tward. Plasma methylome landscape across the spectrum of prostate cancer progression, and differentially methylated region (DMR) based biomarkers of hormonal therapy failure in metastatic hormone-sensitive 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 6023.

Abstract 3479: Modeling epigenetic lesions that cause gliomas

Abstract Epigenetic lesions that disrupt gene regulatory elements and expression are increasingly recognized as potential drivers of human cancers. However, we currently lack the in vitro and in vivo models required to functionally validate such lesions and their tumorigenic impact. Here we model aberrations that arise in Isocitrate Dehydrogenase (IDH) mutant lower-grade gliomas, which exhibit profound DNA hypermethylation as a direct consequence of mutant IDH. DNA hypermethylation may promote gliomagenesis by silencing tumor suppressor genes or, alternatively, by activating proto-oncogenes through disruption of CCCTC-binding factor (CTCF) insulators. CTCF insulator sites define the three-dimensional shape of the genome by dictating the boundaries of topologically associated domains (TADs). Enhancers and promoters can interact when located in the same TAD but are restricted from interacting across different TADs. In IDH mutant gliomas, CpG sites around CTCF binding sites are frequently methylated, effectively compromising CTCF binding and thus TAD organization, allowing for cross-TAD interactions and aberrant activation of genes. We discovered a CTCF insulator downstream of the PDGFRA proto-oncogene that is recurrently disrupted in IDH mutant gliomas. We demonstrate that disruption of the syntenic insulator in mouse oligodendrocyte progenitor cells (OPCs) allows an OPC-specific enhancer to contact and induce Pdgfra, thereby increasing proliferation. In contrast, insulator disruption did not affect Pdgfra expression in neural progenitor cells (NPCs), which lack the enhancer. We also model a second recurrent epigenetic lesion in IDH mutant gliomas, the methylation-dependent silencing of the CDKN2A tumor suppressor. We show that inactivation of Cdkn2a/p19ARF by de novo promoter methylation or mutation drives OPC proliferation and cooperates with Pdgfra insulator loss. Finally, we use lentiviruses to coordinately inactivate the Pdgfra insulator and Cdkn2a in mouse corpus callosum, resulting in low-grade gliomagenesis in vivo with histological features reminiscent of human IDH mutant gliomas. Our study recapitulates recurrent epigenetic lesions in mouse models and demonstrates that the combination of Pdgfra activation and Cdkn2a silencing can transform OPCs in vitro and drive gliomagenesis in vivo. Citation Format: Gilbert J. Rahme, Nauman M. Javed, Kaitlyn L. Puorro, Shouhui Xin, Volker Hovestadt, Sarah E. Johnstone, Bradley E. Bernstein. Modeling epigenetic lesions that cause gliomas [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 3479.