Repression Of Tumor Suppressor Genes Research Articles

Metabolic Signatures and Pathway Rewiring in TET2-Mutated Myeloid Neoplasia

Background. TET2 mutations ( TET2 MT) are the most frequently observed somatic lesions in myeloid neoplasms (MN). TET2 belongs to an Fe2+ and α-ketoglutarate (αKG) dependent DNA-dioxygenase family that progressively oxidizes 5-methyldeoxycytidine (5mC), leading to promoter and enhancer mCpG demethylation essential for efficient transcription. TET2 accounts for the majority of DNA dioxygenase activity in hematopoietic stem and progenitor cells (HSPCs), and following its loss, the resulting accumulation of 5mC leads to a loss of lineage plasticity and transcriptional repression of tumor suppressor genes, thereby establishing a volatile pre-neoplastic state. TET2 is one of the major consumers of αKG, a major cofactor produced by the tricarboxylic acid (TCA) cycle. Therefore, loss of TET2 creates a metabolic perturbation in HSPCs. However, the impact of TET2 loss on the metabolism of leukemic cells and its subsequent effects on malignant evolution remains unclear. Methods. We generated isogenic TET2 -/- ( TET2 ko) leukemic cell models in THP1 and K562 using CRISPR-Cas9 knockout. We then utilized targeted and untargeted metabolic analyses by ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry in these models. We aimed to investigate the intricate metabolic changes associated with TET2 and TET2 MT. To validate our findings, we extended our studies to primary cells derived from MN patients with and without TET2 lesions. We performed extensive cellular metabolism studies using 13C isotope labelled glucose and glutamine. Additionally, levels of 5mC and its oxidation products, namely 5-hydroxymethyldeoxycytidine, 5-formyldeoxycytidine, and 5-carboxydeoxycytidine were documented for correlating epigenetic modifications with TET2 activities. Results. Targeted metabolic analysis uncovered profound impacts of TET2 MT on key metabolic pathways including the TCA cycle, glycolysis, glutamine metabolism, and nucleoside biosynthesis (Figure A). Distinct changes were observed in cells from different lineage backgrounds, suggesting diverse metabolic pathway rewiring specific to cell origin. Despite the divergence among lineages, loss of TET2 created common and unique metabolic signatures, highlighting their crucial role in sustaining the survival and clonal advantages specific to TET2 MT. Representative signature compounds and their relative quantities in TET2 wildtype (WT) and TET2 MT are depicted in Figure A. The targeted metabolic analysis revealed a significantly lower level of glutamine in TET2 MTcells, suggesting a potential preference for exhausting glutamine as a source of energy production. For validation, we conducted 13C metabolic flux analysis (13C-MFA) using glucose (U-13C, 99%) and L-glutamine (U-13C, 99%). 13C-MFA revealed distinct enrichment patterns (Figure B). In the TCA cycle, the dominant isotopologues receiving [U-13C]-glucose were M+2 for citrate and isocitrate. However, for aKG, succinate (SA) and fumarate (FA), the M+2s were significantly lower in TET2 -/- compared to WT, suggesting that the major source for these metabolites were rewired from glycolysis to glutamine metabolism in TET2 -/-. This was confirmed by [U-13C]-glutamine labeling where dominant isotopologues, M+5 for aKG and M+4 for SA, FA and malate, were significantly higher at 24-hour time point in in TET2 -/- compared to WT. Untargeted metabolic analyses from isogenic cells were used to identify metabolic features associated with TET2 loss. These metabolic features were also assayed in primary AML cells with TET2 MT (n=12) and without: RUNX1 (n=20), SF3B1 (n=11), IDH1 (n=10), IDH2 (n=13), FH (n=3), as well as healthy donors as controls (n=6). Based on the molecular features identified in the TET2 ko isogenic cells, MN patients with TET2, IDH1/2, and germline FH mutations clustered more closely, suggesting shared metabolic signatures among these groups. In contrast, patients with RUNX1 and SF3B1 mutations formed separate clusters, demonstrating distinct metabolic profiles associated with these genetic alterations. Conclusion. Our findings provide comprehensive and novel insights into the metabolic consequences of TET2 inactivation in MN. The identified metabolic signatures and rewiring of key pathways may hold promise as potential therapeutic targets and offer opportunities for tailored treatment approaches in TET2MT MN.

Novel Protac-Based EZH2 Degraders Effectively Target EZH2-Driven Lymphoid Malignancies By Abolishing Both Methylation-Dependent and -Independent Functions

Across various cancers, overexpression or mutation of the epigenetic silencer EZH2 correlates with invasive cancer growth, resistance and poor survival outcomes. While the tumorigenic role of EZH2 is largely attributed to the H3K27me3-mediated repression of tumor suppressor genes, reports have also established a methylation-independent role for EZH2 in the transcriptional activation of oncogenes, particularly in aggressive cancers like Natural Killer T-cell Lymphoma (NKTL). Here, traditional EZH2 inhibitors have been ineffective. We hypothesized that targeted depletion of the EZH2 protein will more effectively disrupt oncogenic signals arising from enzyme-independent activities of EZH2. Thus, we embarked on the development of EZH2 Proteolysis Targeting Chimeras (PROTACs) to trigger targeted ubiquitination and proteasome-mediated degradation of EZH2. No clinical EZH2 PROTAC has been reported yet. Our initial efforts focused on the iterative synthesis of a series of PROTAC candidates considering factors such as permeability, solubility and stable ternary complex formation. We developed a library of 53 compounds with various permutations of EZH2- and E3 ligase-binding ligands as well as linker lengths. Through extensive screening and testing of the EZH2 degradation activity via the Nano-Glo HiBiT lytic detection system and immunoblotting, we identified three lead Protac candidates. Compared to the clinical EZH2 inhibitor Tazemetostat (Taz), EZH2 PROTACs, C2911 and C1311 demonstrated higher cytotoxic activity in diffuse large B-cell lymphoma (DLBCL), a cancer in which Taz was evaluated during clinical trial. Importantly, C2911 and C1311 also successfully triggered EZH2 degradation in a dose-dependent manner thereby qualifying for further evaluation in NKTL. Previous work has highlighted a critical role for JAK3-mediated phosphorylation of EZH2 in promoting NKTL proliferation. Treatment of NKTL with C2911 and C1311 leads to a dose and time-dependent EZH2 degradation, loss of cell viability and increased apoptosis. These effects were absent with Taz and Pomalidomide treatments. The concomitant loss of H3K27me3 levels as well as PRC2 complex subunits, SUZ12 and EED suggest effective degradation of the EZH2 protein. We also observed that induction of NKTL cell death required a longer treatment period as compared to DLBCL. This may be potentially ascribed to the longer half-life of the EZH2 protein in NKTL as determined through cycloheximide treatment. In order to validate the mechanism of action of the EZH2 PROTACs, cells were either pretreated with pomalidomide or siRNA targeting cereblon. Indeed, this mitigated PROTAC-induced degradation of the EZH2 protein and rescued cancer cell death. Similarly, inhibiting the activity of the 26S proteasome through bortezomib also prevented EZH2 degradation. Characterization of EZH2 PROTAC-induced protein expression changes through immunoblotting highlighted that there was specific loss of H3K27me3 and H3K27me2 but not H3K27me1. Of note, we detected a concomitant decrease of Jak3 protein expression and STAT5 phosphorylation levels, more pronounced than PRC2 complex subunits. This suggests that the PROTAC may act by disrupting the non-canonical binding of EZH2 to Jak3 in NKTL. Furthermore, gene expression analyses revealed that EZH2 PROTAC treatment more effectively reactivated canonical EZH2 target genes such as MYT1 and ANPEP as compared to Taz. As the JAK-STAT-MYC axis is dysregulated in NK leukemia, we also evaluated the gene expression of c-Myc and c-Myc target gene, E2F1. EZH2 PROTAC treatment reduces the transcription levels of these oncogenes corroborating with loss of c-Myc protein expression. Studying the pharmacokinetics of the lead PROTACs in a mouse model revealed good plasma stability, half-life (> 4 h), and low clearance. Collectively, our data supports a critical role for EZH2 PROTACs in abolishing both the enzyme-dependent and independent activities of EZH2 through protein degradation, resulting in enhanced anti-cancer activity against DLBCL and NKTL. This may potentially benefit other EZH2-driven cancers. Further evaluation of the EZH2 PROTACs for clinical application is in progress. EZH2 PROTACs will next be tested in vivo. An in-depth investigation of the mechanisms stabilizing EZH2 protein levels and underlying PROTAC induced-cell death will be made to identify potential mechanisms of resistance.

EZH2-H3K27me3-mediated silencing of mir-139-5p inhibits cellular senescence in hepatocellular carcinoma by activating TOP2A

BackgroundEpigenetic alterations play an important role in hepatocellular carcinoma (HCC) development. Enhancer of zeste homolog 2 (EZH2) is a well-known epigenetic modifier that functions as an oncogene in tumors by promoting the H3K27me3-mediated transcriptional repression of tumor suppressor genes. “Senescent cells” has been proposed as a possible core component of the hallmarks of cancer conceptualization. Induction of cell senescence and targeted elimination of these senescent tumor cells are new strategies for tumor therapy. However, the role of EZH2 in regulating cellular senescence remains poorly understood.MethodsBioinformatics analyses suggested that EZH2 and DNA topoisomerase II alpha (TOP2A) are coexpressed in tumors, including HCC. Kyoto Encyclopedia of Genes and Genome (KEGG) pathway enrichment analyses and gene set enrichment analyses (GSEA) suggests a correlation of EZH2 and TOP2A expression with cellular senescence in HCC. MicroRNA (miRNA) inhibitor and mimics, siRNA, PLKO-shRNA, and plenti6.3-miR-139 were used to upregulate or downregulate the expression of target genes. CCK8, EdU, clone formation, and senescence-associated β-galactosidase (SA-β-gal) staining assays were performed to assess cell proliferation and cellular senescence phenotypes. Dual-luciferase reporter and chromatin immunoprecipitation assays were performed to investigate the targeted binding and inhibition of TOP2A 3′ untranslated region (UTR) by miR-139-5p and the DNA enrichment of miR139-5p by EZH2 and H3K27me3. BALB/c nude mice were used to establish a xenograft tumor model and verify the phenotypes upon EZH2 and TOP2A silencing and miR-139 overexpression in vivo. In addition, tissue microarrays were used to analyze the expression patterns and correlations among EZH2, TOP2A, and miR-139-5p expression in HCC.ResultsBioinformatics analysis revealed that EZH2 and TOP2A are coexpressed in HCC. In vitro gain- and loss-of-function experiments showed that inhibition of EZH2 and TOP2A induces cellular senescence and inhibits proliferation of HCC cells. In vivo tumorigenesis assays indicated that EZH2 and TOP2A knockdown inhibits tumorigenesis by inducing cellular senescence. Mechanistically, EZH2 promotes TOP2A expression by regulating the H3K27me3-mediated epigenetic silencing of miR-139-5p. TOP2A is a direct target of miR-139-5p, and inhibition of miR-139-5p can reverse the promotion by EZH2 of TOP2A expression. The overexpression of miR-139-5p induces cellular senescence and inhibits proliferation of HCC cells both in vitro and in vivo. Clinically, expression of EZH2 and TOP2A are higher in HCC tissues than in normal tissues, and this high coexpression indicates a worse outcome of patients with HCC. Moreover, expression of EZH2 and TOP2A is significantly correlated with tumor differentiation grade, tumor invasion, and TNM stage in HCC. miR-139-5p expression is lower in HCC tumors than in normal tissues and is correlated with better prognosis of HCC patients.ConclusionsOur study revealed the role of the EZH2/miR-139-5p/TOP2A axis in regulating cellular senescence and cell proliferation in HCC, enriching the molecular mechanisms of EZH2-mediated epigenetic regulation in HCC. Therefore, our results provide insight into the therapeutic potential of targeting EZH2 to induce cellular senescence and then destroy senescent cells for HCC.Graphic

EZH2 Cooperates with BRD4-NUT to Drive NUT Carcinoma Growth by Silencing Key Tumor Suppressor Genes.

Repression of tumor suppressor genes, including CDKN2A, by EZH2 provides a mechanistic rationale for combining EZH2 and BET inhibitors for the clinical treatment of NUT carcinoma. See related commentary by Kazansky and Kentsis, p. 3827.

Toblerone: detecting exon deletion events in cancer using RNA-seq.

Cancer is driven by mutations of the genome that can result in the activation of oncogenes or repression of tumour suppressor genes. In acute lymphoblastic leukemia (ALL) focal deletions in IKAROS family zinc finger 1 (IKZF1) result in the loss of zinc-finger DNA-binding domains and a dominant negative isoform that is associated with higher rates of relapse and poorer patient outcomes. Clinically, the presence of IKZF1 deletions informs prognosis and treatment options. In this work we developed a method for detecting exon deletions in genes using RNA-seq with application to IKZF1. We developed a pipeline that first uses a custom transcriptome reference consisting of transcripts with exon deletions. Next, RNA-seq reads are mapped using a pseudoalignment algorithm to identify reads that uniquely support deletions. These are then evaluated for evidence of the deletion with respect to gene expression and other samples. We applied the algorithm, named Toblerone, to a cohort of 99 B-ALL paediatric samples including validated IKZF1 deletions. Furthermore, we developed a graphical desktop app for non-bioinformatics users that can quickly and easily identify and report deletions in IKZF1 from RNA-seq data with informative graphical outputs.

TBX2 acts as a potent transcriptional silencer of tumour suppressor genes through interaction with the CoREST complex to sustain the proliferation of breast cancers.

Chromosome 17q23 amplification occurs in 20% of primary breast tumours and is associated with poor outcome. The TBX2 gene is located on 17q23 and is often over-expressed in this breast tumour subset. TBX2 is an anti-senescence gene, promoting cell growth and survival through repression of Tumour Suppressor Genes (TSGs), such as NDRG1 and CST6. Previously we found that TBX2 cooperates with the PRC2 complex to repress several TSGs, and that PRC2 inhibition restored NDRG1 expression to impede cellular proliferation. Here, we now identify CoREST proteins, LSD1 and ZNF217, as novel interactors of TBX2. Genetic or pharmacological targeting of CoREST emulated TBX2 loss, inducing NDRG1 expression and abolishing breast cancer growth in vitro and in vivo. Furthermore, we uncover that TBX2/CoREST targeting of NDRG1 is achieved by recruitment of TBX2 to the NDRG1 promoter by Sp1, the abolishment of which resulted in NDRG1 upregulation and diminished cancer cell proliferation. Through ChIP-seq we reveal that 30% of TBX2-bound promoters are shared with ZNF217 and identify novel targets repressed by TBX2/CoREST; of these targets a lncRNA, LINC00111, behaves as a negative regulator of cell proliferation. Overall, these data indicate that inhibition of CoREST proteins represents a promising therapeutic intervention for TBX2-addicted breast tumours.

Aberrant promoter methylation contributes to LRIG1 silencing in basal/triple-negative breast cancer

BackgroundLRIG1, the founding member of the LRIG (leucine-rich repeat and immunoglobulin-like domain) family of transmembrane proteins, is a negative regulator of receptor tyrosine kinases and a tumour suppressor. Decreased LRIG1 expression is consistently observed in cancer, across diverse tumour types, and is linked to poor patient prognosis. However, mechanisms by which LRIG1 is repressed are not fully understood. Silencing of LRIG1 through promoter CpG island methylation has been reported in colorectal and cervical cancer but studies in breast cancer remain limited.MethodsIn silico analysis of human breast cancer patient data were used to demonstrate a correlation between DNA methylation and LRIG1 silencing in basal/triple-negative breast cancer, and its impact on patient survival. LRIG1 gene expression, protein abundance, and methylation enrichment were examined by quantitative reverse-transcription PCR, immunoblotting, and methylation immunoprecipitation, respectively, in breast cancer cell lines in vitro. We examined the impact of global demethylation on LRIG1 expression and methylation enrichment using 5-aza-2’-deoxycytidine. We also examined the effects of targeted demethylation of the LRIG1 CpG island, and transcriptional activation of LRIG1 expression, using the RNA guided deadCas9 transactivation system.ResultsAcross breast cancer subtypes, LRIG1 expression is lowest in the basal/triple-negative subtype so we investigated whether differential methylation may contribute to this. Indeed, we find that LRIG1 CpG island methylation is most prominent in basal/triple-negative cell lines and patient samples. Use of the global demethylating agent 5-aza-2’-deoxycytidine decreases methylation leading to increased LRIG1 transcript expression in basal/triple-negative cell lines, while having no effect on LRIG1 expression in luminal/ER-positive cell lines. Using a CRISPR/deadCas9 (dCas9)-based targeting approach, we demonstrate that TET1-mediated demethylation (Tet1-dCas9) along with VP64-mediated transcriptional activation (VP64-dCas9) at the CpG island, increased endogenous LRIG1 expression in basal/triple-negative breast cancer cells, without transcriptional upregulation at predicted off-target sites. Activation of LRIG1 by the dCas9 transactivation system significantly increased LRIG1 protein abundance, reduced site-specific methylation, and reduced cancer cell viability. Our findings suggest that CRISPR-mediated targeted activation may be a feasible way to restore LRIG1 expression in cancer.ConclusionsOur study contributes novel insight into mechanisms which repress LRIG1 in triple-negative breast cancer and demonstrates for the first time that targeted de-repression of LRIG1 in cancer cells is possible. Understanding the epigenetic mechanisms associated with repression of tumour suppressor genes holds potential for the advancement of therapeutic approaches.

Epigenetic Regulation of Angiogenesis in Development and Tumors Progression: Potential Implications for Cancer Treatment.

Angiogenesis is a multi-stage process of new blood vessel development from pre-existing vessels toward an angiogenic stimulus. The process is essential for tissue maintenance and homeostasis during embryonic development and adult life as well as tumor growth. Under normal conditions, angiogenesis is involved in physiological processes, such as wound healing, cyclic regeneration of the endometrium, placental development and repairing certain cardiac damage, in pathological conditions, it is frequently associated with cancer development and metastasis. The control mechanisms of angiogenesis in carcinogenesis are tightly regulated at the genetic and epigenetic level. While genetic alterations are the critical part of gene silencing in cancer cells, epigenetic dysregulation can lead to repression of tumor suppressor genes or oncogene activation, becoming an important event in early development and the late stages of tumor development, as well. The global alteration of the epigenetic spectrum, which includes DNA methylation, histone modification, chromatin remodeling, microRNAs, and other chromatin components, is considered one of the hallmarks of cancer, and the efforts are concentrated on the discovery of molecular epigenetic markers that identify cancerous precursor lesions or early stage cancer. This review aims to highlight recent findings on the genetic and epigenetic changes that can occur in physiological and pathological angiogenesis and analyze current knowledge on how deregulation of epigenetic modifiers contributes to tumorigenesis and tumor maintenance. Also, we will evaluate the clinical relevance of epigenetic markers of angiogenesis and the potential use of “epi-drugs” in modulating the responsiveness of cancer cells to anticancer therapy through chemotherapy, radiotherapy, immunotherapy and hormone therapy as anti-angiogenic strategies in cancer.

Abstract 1128: Selective, novel, small molecule PRMT5 inhibitors for treatment of cancer

Abstract Introduction: Arginine methylation deregulation in cancer has been well studied and PRMT5 which modulates dimethylation of arginine has emerged as an attractive therapeutic strategy in various cancer types, including lung cancer, lymphoma, glioblastoma, pancreatic cancers, etc. PRMT5 is over-expressed in multiple cancers leading to repression of tumor suppressor genes and genetic studies have identified it is a validated target in lymphoma. Recently, dysregulation of the splicing machinery in cancers has been identified to be one of the therapeutic vulnerabilities for PRMT5 inhibition, especially in glioblastoma. Therefore, inhibitors selectively targeting PRMT5 could be of high clinical value, especially in cancers with defects in spliceosome machinery. Methods: Rational design and structure based drug design were used to identify novel PRMT5 inhibitors. To assess in vitro potency, flash plate based activity assay was used. Cell based activity of these inhibitors was assessed by measuring the symmetrical dimethylation of known cellular protein SmD3 by ELISA and Western blotting. Long term cell proliferation assays were used to assess the functional effect of PRMT5 inhibition. Tumor growth inhibition was measured in orthotopic glioblastoma model in mice. Results: One of the lead PRMT5 inhibitors had an in vitro potency of 3 nM in the biochemical assay which translated well in the cell based SDMA ELISA where the EC50 was <10 nM. Anti-proliferative activity of this molecule in lymphoma, leukaemia, SCLC, pancreatic, lung and glioblastoma cell lines ranged from ~16 nM to 1035 nM. This molecule showed acceptable in vitro ADME properties in terms of aqueous solubility and metabolic stability and excellent oral bioavailability in rodent pharmacokinetics. In Z-138 mantle cell lymphoma xenograft model, oral administration of the lead compound at 50 mg/kg resulted in strong and complete (~95 %) tumour growth inhibition and with a concomitant complete inhibition of SDMA. The lead compound was well tolerated with no reduction in body weight at the tested doses. Interestingly, this molecule showed excellent brain exposure sufficient to achieve target engagement for 10h and significant tumour growth inhibition of orthotopic brain tumors by oral dosing. Repeat dose non-GLP study in rodents clearly demonstrated the safety of this molecule. Conclusion: Given the therapeutic importance of PRMT5 in glioblastoma and other lymphomas, this molecule will be extremely valuable in treating these cancers both as a standalone therapy and in combination with other standard of care agents. Citation Format: Dhanalakshmi Sivanandhan, Sridharan Rajagopal, Naveen Sadhu M, Chandru Gajendran, Saravanan Vadivelu, Natarajan Tamizharasan, Indu N. Swamy, Santhosh Viswakarma, Amir Siddiqui, Saif wahid, Mohammed Zainuddin, Rudresh G, Prashanthi Daram, Ramchandraiah Gosu, Dinesh Tiagaraj, Shivani Garapaty, Sreekala Nair, Namratha Kapoor. Selective, novel, small molecule PRMT5 inhibitors for treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1128.

Anticancer Therapy with HDAC Inhibitors: Mechanism-Based Combination Strategies and Future Perspectives.

Simple SummaryBeyond mutations, epigenetic changes have been described as drivers for cancer as well. While leaving the overall DNA structure intact, they can be responsible for tumor malignancy by mediating the transcriptional upregulation of oncogenes. This provides the basis for “epigenetic therapies” in cancer. Histone deacetylases (HDACs) are major players in epigenetic reprogramming. HDAC inhibitors (HDACis), either with broad-spectrum activity on various HDAC isoforms or with specific subtype specificity, have shown promising anticancer efficacies. The tremendous number of genes potentially affected creates the possibility for the parallel targeting of multiple disease-relevant pathways. Here, we give a comprehensive overview of various preclinical and clinical studies on HDACis. A particular focus is placed on the detailed description of promising strategies based on the combination of HDACis with other drugs. This also includes the development of new bifunctional inhibitors as well as novel approaches for HDAC degradation, rather than inhibition, via PROteolysis-TArgeting Chimeras (PROTACs).The increasing knowledge of molecular drivers of tumorigenesis has fueled targeted cancer therapies based on specific inhibitors. Beyond “classic” oncogene inhibitors, epigenetic therapy is an emerging field. Epigenetic alterations can occur at any time during cancer progression, altering the structure of the chromatin, the accessibility for transcription factors and thus the transcription of genes. They rely on post-translational histone modifications, particularly the acetylation of histone lysine residues, and are determined by the inverse action of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Importantly, HDACs are often aberrantly overexpressed, predominantly leading to the transcriptional repression of tumor suppressor genes. Thus, histone deacetylase inhibitors (HDACis) are powerful drugs, with some already approved for certain hematological cancers. Albeit HDACis show activity in solid tumors as well, further refinement and the development of novel drugs are needed. This review describes the capability of HDACis to influence various pathways and, based on this knowledge, gives a comprehensive overview of various preclinical and clinical studies on solid tumors. A particular focus is placed on strategies for achieving higher efficacy by combination therapies, including phosphoinositide 3-kinase (PI3K)-EGFR inhibitors and hormone- or immunotherapy. This also includes new bifunctional inhibitors as well as novel approaches for HDAC degradation via PROteolysis-TArgeting Chimeras (PROTACs).

Global Genome Demethylation Causes Transcription-Associated DNA Double Strand Breaks in HPV-Associated Head and Neck Cancer Cells

Simple SummaryHigh levels of global genome methylation in HPV-associated head and neck cancer prompted us to explore demethylation as a potential treatment by determining mechanisms of its toxicity in HPV-positive head and neck cancer cells. Previously, we reported that demethylating drug 5-azaC stabilizes p53 and reduces the expression of HPV genes and matrix metalloproteinases in HPV+ head and neck cancer cells and tumors from patients enrolled in a 5-azaC window clinical trial. Here, we extended our understanding of toxicity caused by global demethylation in HPV-associated head and neck cancer cells by finding that 5-azaC treatment results in formation of DNA double strand breaks that depend on transcription and replication.High levels of DNA methylation at CpG loci are associated with transcriptional repression of tumor suppressor genes and dysregulation of DNA repair genes. Human papilloma virus (HPV)-associated head and neck squamous cell carcinomas (HNSCC) have high levels of DNA methylation and methylation has been associated with dampening of an innate immune response in virally infected cells. We have been exploring demethylation as a potential treatment in HPV+ HNSCC and recently reported results of a window clinical trial showing that HNSCCs are particularly sensitive to demethylating agent 5-azacytidine (5-aza). Mechanistically, sensitivity is partially due to downregulation of HPV genes expression and restoration of tumor suppressors p53 and Rb. Here, for the first time, we show that 5-azaC treatment of HPV+ HNSCC induces replication and transcription-associated DNA double strand breaks (DSBs) that occur preferentially at demethylated genomic DNA. Blocking replication or transcription prevented formation of DNA DSBs and reduced sensitivity of HPV-positive head and neck cancer cells to 5-azaC, demonstrating that both replication and active transcription are required for formation of DSBs associated with 5-azaC.

Polycomb repressor complex 2 function in breast cancer (Review)

Epigenetic modifications are important contributors to the regulation of genes within the chromatin. The poly-comb repressive complex 2 (PRC2) is a multi-subunit protein complex that is involved in silencing gene expression through the trimethylation of lysine 27 at histone 3 (H3K27me3). The dysregulation of this modification has been associated with tumorigenicity through the increased repression of tumour suppressor genes via condensing DNA to reduce access to the transcription start site (TSS) within tumor suppressor gene promoters. In the present review, the core proteins of PRC2, as well as key accessory proteins, will be described. In addition, mechanisms controlling the recruitment of the PRC2 complex to H3K27 will be outlined. Finally, literature identifying the role of PRC2 in breast cancer proliferation, apoptosis and migration, including the potential roles of long non-coding RNAs and the miR-200 family will be summarized as will the potential use of the PRC2 complex as a therapeutic target.

Y-box binding protein 1 acts as a negative regulator of stearoyl CoA desaturase 1 in clear cell renal cell carcinoma.

Y-box binding protein 1 (YB-1) is a regulatory protein associated with oncogenesis and poor prognosis in patients with cancer. In the cell, YB-1 functions as a DNA and RNA binding protein that promotes or suppresses expression of target genes. The cancer-promoting activity of YB-1 is mediated through its activation of oncogenes and repression of tumor suppressor genes. Lipogenic enzyme stearoyl-CoA desaturase (SCD1) drives the production of endogenous monounsaturated fatty acids (MUFAs) in cells and protects against toxic buildup of saturated fatty acids. Clear cell renal cell carcinoma (ccRCC) is often characterized by aberrantly high SCD1 expression and cytosolic accumulation of unsaturated fatty acids. In the present study, a proteomics screen of cells treated with inhibitors of SCD1 supported a potential relationship between YB-1 and SCD1. It was revealed that the presence of MUFAs led to increased protein synthesis and increased expression of high molecular weight forms of YB-1 in ccRCC cells, but not in non-tumorigenic cells. Ectopic expression of YB-1 led to decreased expression levels of SCD1 protein and mRNA in ccRCC cell lines. Conversely, targeted knockdown of YB-1 increased SCD1 mRNA abundance. Analysis of ccRCC patient data from The Cancer Proteome Atlas database showed YB-1 expression was negatively associated with survival, whereas SCD1 was associated with improved survival. These data suggested an antagonistic relationship between YB-1 and SCD1 that may influence survival of patients with ccRCC.

Abstract 2915: Preclinical characterization of PRT543, a potent and selective inhibitor of protein arginine methyltransferase 5 (PRMT5), with broad antitumor activity in in vitro and in vivo models

Abstract Protein arginine methyltransferase 5 (PRMT5) is the major type II PRMT that catalyzes the formation of symmetrical dimethyl arginine (SDMA) on protein substrates and plays important roles in various cellular pathways including tumorigenesis. PRMT5 can methylate histones H3 and H4, thereby leading to repression of tumor suppressor genes at specific loci. PRMT5 also regulates gene expression on a global level by methylating and altering the function of transcription factors such as p53, E2F-1, NF-κB and others. Additionally, PRMT5 has been shown to interact with members of the spliceosome complex and regulate pre-mRNA processing of key target genes. In this study, we describe the in vitro and in vivo activity of PRT543, a novel, potent and selective inhibitor of PRMT5, in hematological and solid tumor models. In a scintillation proximity based radiometric assay, PRT543 inhibited the methyltransferase activity of the PRMT5/MEP50 complex with an IC50 of 10.8 nM. It was also highly selective against a panel of 36 other methyltransferases. PRT543 robustly inhibited cell proliferation in a panel of >50 cancer cell lines, representing both solid tumors and cancers of hematological origin, in a concentration-dependent manner with IC50 values ranging from 10 - 1000 nM. This anti-proliferative activity correlated with a reduction of symmetrically dimethylated SmD3 protein, a known PRMT5 substrate, demonstrating effective inhibition of PRMT5 enzymatic activity in cells. PRT543 demonstrated good oral bioavailability and favorable pharmacokinetic properties. Oral administration of this compound led to significant tumor growth inhibition in several subcutaneous mouse xenograft models including mantle cell lymphoma (Granta-519, Z-138), acute myeloid leukemia (MV4-11, SET2, HEL), small cell lung cancer (NCI-H1048) and bladder cancer (5637) at well-tolerated doses. This in vivo activity was accompanied by a corresponding decrease in symmetrically dimethylated SmD3 in tumor tissue collected from the treated animals. PRT543 was also tested in combination with other approved targeted therapies both in vitro and in vivo. Combining PRT543 with the BCL2 inhibitor, venetoclax, revealed a potent synergistic interaction in models of leukemia and lymphoma. Further ex vivo testing in genetically-defined primary patient tumor samples is ongoing. PRT543 is currently under evaluation in a Phase I clinical trial in patients with advanced solid tumors and hematological malignancies (NCT03886831). Citation Format: Neha Bhagwat, Yang Zhang, Hong Lin, Min Wang, Dave Rominger, Tom Emm, Divya Chugani-Mahtani, Dimitrios Angelis, Rupa Shetty, Raul Leal, William Gowen-MacDonald, Alexander Grego, Juan Luengo, Taghi Manshouri, Friederike Pastore, Ross L. Levine, Srdan Verstovsek, Bruce Ruggeri, Peggy Scherle, Kris Vaddi. Preclinical characterization of PRT543, a potent and selective inhibitor of protein arginine methyltransferase 5 (PRMT5), with broad antitumor activity in in vitro and in vivo models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2915.

Protein lysine 43 methylation by EZH1 promotes AML1-ETO transcriptional repression in leukemia

The oncogenic fusion protein AML1-ETO retains the ability of AML1 to interact with the enhancer core DNA sequences, but blocks AML1-dependent transcription. Previous studies have shown that post-translational modification of AML1-ETO may play a role in its regulation. Here we report that AML1-ETO-positive patients, with high histone lysine methyltransferase Enhancer of zeste homolog 1 (EZH1) expression, show a worse overall survival than those with lower EZH1 expression. EZH1 knockdown impairs survival and proliferation of AML1-ETO-expressing cells in vitro and in vivo. We find that EZH1 WD domain binds to the AML1-ETO NHR1 domain and methylates AML1-ETO at lysine 43 (Lys43). This requires the EZH1 SET domain, which augments AML1-ETO-dependent repression of tumor suppressor genes. Loss of Lys43 methylation by point mutation or domain deletion impairs AML1-ETO-repressive activity. These findings highlight the role of EZH1 in non-histone lysine methylation, indicating that cooperation between AML1-ETO and EZH1 and AML1-ETO site-specific lysine methylation promote AML1-ETO transcriptional repression in leukemia.

Epigenetic Regulation of TRAIL Signaling: Implication for Cancer Therapy.

One of the main characteristics of carcinogenesis relies on genetic alterations in DNA and epigenetic changes in histone and non-histone proteins. At the chromatin level, gene expression is tightly controlled by DNA methyl transferases, histone acetyltransferases (HATs), histone deacetylases (HDACs), and acetyl-binding proteins. In particular, the expression level and function of several tumor suppressor genes, or oncogenes such as c-Myc, p53 or TRAIL, have been found to be regulated by acetylation. For example, HATs are a group of enzymes, which are responsible for the acetylation of histone proteins, resulting in chromatin relaxation and transcriptional activation, whereas HDACs by deacetylating histones lead to chromatin compaction and the subsequent transcriptional repression of tumor suppressor genes. Direct acetylation of suppressor genes or oncogenes can affect their stability or function. Histone deacetylase inhibitors (HDACi) have thus been developed as a promising therapeutic target in oncology. While these inhibitors display anticancer properties in preclinical models, and despite the fact that some of them have been approved by the FDA, HDACi still have limited therapeutic efficacy in clinical terms. Nonetheless, combined with a wide range of structurally and functionally diverse chemical compounds or immune therapies, HDACi have been reported to work in synergy to induce tumor regression. In this review, the role of HDACs in cancer etiology and recent advances in the development of HDACi will be presented and put into perspective as potential drugs synergizing with TRAIL’s pro-apoptotic potential.

JAK2 regulates mismatch repair protein-mediated epigenetic alterations in response to oxidative damage.

At sites of chronic inflammation epithelial cells undergo aberrant DNA methylation that contributes to tumorigenesis. Inflammation is associated with an increase in reactive oxygen species (ROS) that cause oxidative DNA damage, which has also been linked to epigenetic alterations. We previously demonstrated that in response to ROS, mismatch repair proteins MSH2 and MSH6 recruit epigenetic silencing proteins DNA methyltransferase 1 (DNMT1) and polycomb repressive complex 2 (PRC2) members to sites of DNA damage, resulting in transcriptional repression of tumor suppressor genes (TSGs). However, it was unclear what signal is unique to ROS that results in the chromatin binding of MSH2 and MSH6. Herein, we demonstrate that in response to hydrogen peroxide (H2 O2 ), JAK2 localizes to the nucleus and interacts with MSH2 and MSH6. Inhibition or knockdown of JAK2 reduces the H2 O2 -induced chromatin interaction of MSH2, MSH6, DNMT1, and PRC2 members, reduces H2 O2 -induced global increase in trimethylation of lysine 27 of histone H3 (H3K27me3), and abrogates oxidative damage-induced transcriptional repression of candidate TSGs. Moreover, JAK2 mRNA expression is associated with CpG island methylator phenotype (CIMP) status in human colorectal cancer. Our findings provide novel insight into the connection between kinase activation and epigenetic alterations during oxidative damage and inflammation. Environ. Mol. Mutagen. 60:308-319, 2019. © 2018 Wiley Periodicals, Inc.

Increased EZH2 expression during the adenoma-carcinoma sequence in colorectal cancer.

The adenoma-carcinoma sequence, the sequential mutation and deletion of various genes by which colorectal cancer progresses, is a well-established and accepted concept of colorectal cancer carcinogenesis. Proteins of the polycomb repressive complex 2 (PRC2) function as transcriptional repressors by trimethylating histone H3 at lysine 27; the activity of this complex is essential for cell proliferation and differentiation. The histone methyltransferase enhancer of zeste homolog 2 (EZH2), an essential component of PRC2, is associated with the transcriptional repression of tumor suppressor genes. EZH2 expression has previously been reported to increase with the progression of pancreatic intraductal papillary mucinous neoplasm. Thus, we hypothesized that EZH2 expression also increases during the adenoma-carcinoma sequence of colorectal cancer. The present study investigated changes in EZH2 expression during the colorectal adenoma-carcinoma sequence. A total of 47 patients with colorectal adenoma, 20 patients with carcinoma in adenoma and 43 patients with colorectal carcinoma who underwent surgical or endoscopic resection were enrolled in this study. Non-cancerous tissue from the clinical specimens was also examined. The association between EZH2 expression, pathology and expression of tumor suppressor genes during colorectal carcinogenesis were analyzed. Each specimen was immunohistochemically stained for EZH2, proliferation marker protein Ki-67 (Ki-67), cyclin-dependent kinase inhibitor (CDKN) 1A (p21), CDKN1B (p27) and CDKN2A (p16). Total RNA was extracted from formalin-fixed paraffin-embedded blocks and reverse transcription-quantitative polymerase chain reaction analysis of these genes was performed. Ki-67 and EZH2 expression scores increased significantly during the progression of normal mucosa to adenoma and carcinoma (P=0.009), and EZH2 expression score was positively associated with Ki-67 expression score (P=0.02). Conversely, p21 mRNA and protein expression decreased significantly, whereas expression of p27 and p16 did not change significantly. During the carcinogenesis sequence from normal mucosa to adenoma and carcinoma, EZH2 expression increased and p21 expression decreased significantly. EZH2 may therefore contribute to the development of colorectal cancer from adenoma via suppression of p21.

Abstract 5339: Epigenetic silencing of ANGPTL4 in tumor tissues and its overexpression in the urothelial carcinoma microenvironment

Abstract Urothelial carcinoma (UC) carcinogenesis has been hypothesized to occur through epigenetic repression of tumor suppressor genes (TSGs). By qRT-PCR array, we found that one potential TSG, ANGPTL4, was expressed at very low levels in all UC cell lines we examined. Previous studies had demonstrated that ANGPTL4 is highly expressed in some cancers, but downregulated, by DNA methylation, in others. Consequently, due to these seemingly conflicting functions in distinct cancers, the precise role of ANGPTL4 in the etiology of UC remains unclear. In this study, using methylation-specific PCR and bisulfite pyrosequencing, we show that ANGPTL4 is transcriptionally repressed by promoter hypermethylation in UC cell lines and primary tumor samples, as compared to adjacent noncancerous bladder urothelium. Functional studies further demonstrated that ectopic expression of ANGPTL4 potently suppressed UC cell proliferation, monolayer colony formation in vitro, and invasion, migration, and xenograft formation in vivo. As ANGPTL4 is a secreted factor, we therefore examined the circulating ANGPTL4 (cir-ANGPTL4) level in UC patients. Surprisingly, cir-ANGPTL4 was significantly higher in plasma samples from UC patients than normal control, suggesting it might be secreted from other cell types. More importantly, exogenous c-terminal fragment of ANGPTL4 (cANGPTL4) could promote cell proliferation and cell migration via activation of signaling through the Erk/FAK axis. We further confirmed that mouse xenograft tumor growth could be promoted by administration of exogenous cANGPTL4. Finally, immunohistochemistry demonstrated that ANGPTL4 was downregulated in tumor cells but overexpressed in tumor-adjacent stromal tissues including muscle and macrophages of muscle-invasive UC tissue samples. In conclusion, our data supports dual roles for ANGPTL4 in urothelial carcinoma progression, either as a tumor suppressor or oncogene, in response to microenvironmental context. Citation Format: Michael WY Chan, Hsiao-Yen Hsieh, Yeong-Chin Jou, Chun-Liang Tung, Yuh-Shyan Tsai, Yuan-Hung Wang, Chen-Lin Chi, Ru-Inn Lin, Shih-Kai Hung, Yu-Ming Chuang, Shu-Fen Wu, Chin Li, Cheng-Huang Shen, Cheng-Da Hsu. Epigenetic silencing of ANGPTL4 in tumor tissues and its overexpression in the urothelial carcinoma microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5339.

Effect of a toxic Microcystis aeruginosa lysate on the mRNA expression of proto-oncogenes and tumor suppressor genes in zebrafish

Cyanobacterial blooms of Microcystis aeruginosa represent a significant risk to the environment and have become a worldwide concern. M. aeruginosa can produce the hepatotoxins microcystins (MCs) with potential for tumor promotion. The present study evaluated the time-dependent effects in the transcription of tumor-related genes in the zebrafish, Danio rerio, exposed to dilutions of a M. aeruginosa lysate containing 3.5 and 54.6 µg L−1 MCs. We used a cultured M. aeruginosa strain, RST 9501, which contains mainly the variant [D-Leu1] MC-LR and originated from the Patos Lagoon Estuary (RS, Brazil). The exposure caused short-term repression of tumor suppressor genes and long-term repression of proto-oncogenes. These responses were more evident for p53 that was repressed with exposure for 6, 24 and 96 h, and fosab and myca that were consistently repressed with exposure for 384 h, when fish were exposed to both M. aeruginosa lysate dilutions, compared to controls (p < 0.05). The suppressor genes, baxa and gadd45α, and the proto-oncogene, junba, were suppressed mainly at 96 h, where both dilutions of the lysate caused repression compared to controls (p < 0.05). The p53 gene was the only gene to be induced; this occurred in fish exposed to lysate containing 3.5 µg L−1 for 384 h. This is the first study to show that M. aeruginosa containing an environmentally relevant concentration of [D-Leu1] MC-LR could cause time-dependent repression of proto-oncogenes and tumor suppressor genes in fish. The results suggest that short-term repression of tumor suppressor genes could participate in the mechanism of tumor promotion caused by M. aeruginosa in fish.