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Metaplastic breast carcinomas (mBrCA) are a highly aggressive subtype of triple-negative breast cancer with histologic evidence of epithelial-to-mesenchymal transition and aberrant differentiation. Inactivation of the tumor suppressor gene cellular communication network factor 6 (CCN6; also known as Wnt1-induced secreted protein 3) is a feature of mBrCAs, and mice with conditional inactivation of Ccn6 in mammary epithelium (Ccn6-KO) develop spindle mBrCAs with epithelial-to-mesenchymal transition. Elucidation of the precise mechanistic details of how CCN6 acts as a tumor suppressor in mBrCA could help identify improved treatment strategies. In this study, we showed that CCN6 interacts with the Wnt receptor FZD8 and coreceptor LRP6 on mBrCA cells to antagonize Wnt-induced activation of β-catenin/TCF-mediated transcription. The histone methyltransferase EZH2 was identified as a β-catenin/TCF transcriptional target in Ccn6-KO mBrCA cells. Inhibiting Wnt/β-catenin/TCF signaling in Ccn6-KO mBrCA cells led to reduced EZH2 expression, decreased histone H3 lysine 27 trimethylation, and deregulation of specific target genes. Pharmacologic inhibition of EZH2 reduced growth and metastasis of Ccn6-KO mBrCA mammary tumors in vivo. Low CCN6 is significantly associated with activated β-catenin and high EZH2 in human spindle mBrCAs compared with other subtypes. Collectively, these findings establish CCN6 as a key negative regulator of a β-catenin/TCF/EZH2 axis and highlight the inhibition of β-catenin or EZH2 as a potential therapeutic approach for patients with spindle mBrCAs.  Significance: CCN6 deficiency drives metaplastic breast carcinoma growth and metastasis by increasing Wnt/β-catenin activation to upregulate EZH2, identifying EZH2 inhibition as a mechanistically guided treatment strategy for this deadly form of breast cancer., (©2024 American Association for Cancer Research.)

Recurrent somatic mutations in the BRG1/BRM-associated factor (BAF) chromatin remodeling complex subunit ARID1A occur frequently in advanced urothelial, endometrial, and ovarian clear cell carcinomas, creating an alternative chromatin state that may be exploited therapeutically. The histone methyltransferase EZH2 has been previously identified as targetable vulnerability in the context of ARID1A mutations. In this study, we describe the discovery of tulmimetostat, an orally available, clinical stage EZH2 inhibitor, and it elucidates the aspects of its application potential in ARID1A mutant tumors. Tulmimetostat administration achieved efficacy in multiple ARID1A mutant bladder, ovarian, and endometrial tumor models and improved cisplatin response in chemotherapy-resistant models. Consistent with its comprehensive and durable level of target coverage, tulmimetostat demonstrated greater efficacy than other PRC2-targeted inhibitors at comparable or lower exposures in a bladder cancer xenograft mouse model. Tulmimetostat mediated extensive changes in gene expression, in addition to a profound reduction in global H3K27me3 levels in tumors. Phase I clinical pharmacokinetic and pharmacodynamic data indicated that tulmimetostat exhibits durable exposure and profound target engagement. Importantly, a tulmimetostat controlled gene expression signature identified in whole blood from a cohort of 32 patients with cancer correlated with tulmimetostat exposure, representing a pharmacodynamic marker for the assessment of target coverage for PRC2-targeted agents in the clinic. Collectively, these data suggest that tulmimetostat has the potential to achieve clinical benefit in solid tumors as a monotherapy but also in combination with chemotherapeutic agents, and may be beneficial in various indications with recurrent ARID1A mutations. Significance: The EZH2 inhibitor tulmimetostat achieves comprehensive target inhibition in ARID1A mutant solid tumor models and cancer patients that can be assessed with a pharmacodynamic gene signature in peripheral blood., (©2024 The Authors; Published by the American Association for Cancer Research.)

Summary: In this issue, a study by Kazansky and colleagues explored resistance mechanisms after EZH2 inhibition in malignant rhabdoid tumors (MRT) and epithelioid sarcomas (ES). The study identified genetic alterations in EZH2 itself, along with alterations that converge on RB1-E2F-mediated cell-cycle control, and demonstrated that inhibition of cell-cycle kinases, such as Aurora Kinase B (AURKB) could bypass EZH2 inhibitor resistance to enhance treatment efficacy. See related article by Kazansky et al., p. 965 (6)., (©2024 American Association for Cancer Research.)

Over the past decade, studies have increasingly shed light on a reciprocal relationship between cellular metabolism and cell fate, meaning that a cell's lineage both drives and is governed by its specific metabolic features. A recent study by Zhang and colleagues, published in Cell Metabolism, describes a novel metabolic-epigenetic regulatory axis that governs lineage identity in triple-negative breast cancer (TNBC). Among the key findings, the authors demonstrate that the metabolic enzyme pyruvate kinase M2 (PKM2) directly binds to the histone methyltransferase enhancer of zeste homolog 2 (EZH2) in the nucleus to silence expression of a set of genes that includes the mitochondrial carnitine transporter SLC16A9. Perturbation of this metabolic-epigenetic regulatory mechanism induces a metabolic shift away from glycolysis and toward fatty acid oxidation. The ensuing influx of carnitine facilitates the deposition of the activating epigenetic mark H3K27Ac onto the promoter of GATA3, driving a committed luminal lineage state. Importantly, this metabolic-epigenetic axis represents a potentially targetable vulnerability for the treatment of TNBC, a subtype that currently lacks effective therapeutic strategies. These findings lend further support for the paradigm shift underlying our understanding of cancer metabolism: that a cellular fuel source functions not only to provide energy but also to direct the epigenetic regulation of cell fate., (©2024 American Association for Cancer Research.)

NUT carcinoma is an aggressive carcinoma driven by the BRD4-NUT fusion oncoprotein, which activates chromatin to promote expression of progrowth genes. BET bromodomain inhibitors (BETi) are a promising treatment for NUT carcinoma that can impede BRD4-NUT's ability to activate genes, but the efficacy of BETi as monotherapy is limited. Here, we demonstrated that enhancer of zeste homolog 2 (EZH2), which silences genes through establishment of repressive chromatin, is a dependency in NUT carcinoma. Inhibition of EZH2 with the clinical compound tazemetostat potently blocked growth of NUT carcinoma cells. Epigenetic and transcriptomic analysis revealed that tazemetostat reversed the EZH2-specific H3K27me3 silencing mark and restored expression of multiple tumor suppressor genes while having no effect on key oncogenic BRD4-NUT-regulated genes. Indeed, H3K27me3 and H3K27ac domains were found to be mutually exclusive in NUT carcinoma cells. CDKN2A was identified as the only gene among all tazemetostat-derepressed genes to confer resistance to tazemetostat in a CRISPR-Cas9 screen. Combined inhibition of EZH2 and BET synergized to downregulate cell proliferation genes, resulting in more pronounced growth arrest and differentiation than either inhibitor alone. In preclinical models, combined tazemetostat and BETi synergistically blocked tumor growth and prolonged survival of NUT carcinoma-xenografted mice, with complete remission without relapse in one cohort. Identification of EZH2 as a dependency in NUT carcinoma substantiates the reliance of NUT carcinoma tumor cells on epigenetic dysregulation of functionally opposite, yet highly complementary, chromatin regulatory pathways to maintain NUT carcinoma growth., Significance: 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., (©2023 American Association for Cancer Research.)

In a recent study, Rialdi and colleagues identified a specific vulnerability in β-catenin mutant hepatocellular carcinoma (HCC) via EZH2-mediated suppression of WNT signaling and revealed the selective anti-HCC activity of WNTinib, a chemical derivative of regorafenib and sorafenib in targeting this vulnerability. Their discoveries highlight the role of EZH2 in modulating WNT signaling and suggest an implication of WNTinihb as a small-molecule inhibitor for the treatment of HCC with activated WNT/β-catenin., (©2023 American Association for Cancer Research.)

The immunosuppressive tumor microenvironment in some cancer types, such as luminal breast cancer, supports tumor growth and limits therapeutic efficacy. Identifying approaches to induce an immunostimulatory environment could help improve cancer treatment. Here, we demonstrate that inhibition of cancer-intrinsic EZH2 promotes antitumor immunity in estrogen receptor α-positive (ERα+) breast cancer. EZH2 is a component of the polycomb-repressive complex 2 (PRC2) complex, which catalyzes trimethylation of histone H3 at lysine 27 (H3K27me3). A 53-gene PRC2 activity signature was closely associated with the immune responses of ERα+ breast cancer cells. The stimulatory effects of EZH2 inhibition on immune surveillance required specific activation of type I IFN signaling. Integrative analysis of PRC2-repressed genes and genome-wide H3K27me3 landscape revealed that type I IFN ligands are epigenetically silenced by H3K27me3. Notably, the transcription factor STAT2, but not STAT1, mediated the immunostimulatory functions of type I IFN signaling. Following EZH2 inhibition, STAT2 was recruited to the promoters of IFN-stimulated genes even in the absence of the cytokines, suggesting the formation of an autocrine IFN-STAT2 axis. In patients with luminal breast cancer, high levels of EZH2 and low levels of STAT2 were associated with the worst antitumor immune responses. Collectively, this work paves the way for the development of an effective therapeutic strategy that may reverse immunosuppression in cancer., Significance: Inhibition of EZH2 activates a type I IFN-STAT2 signaling axis and provides a therapeutic strategy to stimulate antitumor immunity and therapy responsiveness in immunologically cold luminal breast cancer., (©2022 American Association for Cancer Research.)

Medulloblastoma has been categorized into four subgroups based on genetic, epigenetic, and transcriptional profiling. Radiation is used for treating medulloblastoma regardless of the subgroup. A better understanding of the molecular pathways determining radiotherapy response could help improve medulloblastoma treatment. Here, we investigated the role of the EZH2 (enhancer of zeste 2 polycomb repressive complex 2 subunit)-dependent histone H3K27 trimethylation in radiotherapy response in medulloblastoma. The tumors in 47.2% of patients with group 3 and 4 medulloblastoma displayed H3K27me3 deficiency. Loss of H3K27me3 was associated with a radioresistant phenotype, high relapse rates, and poor overall survival. In H3K27me3-deficient medulloblastoma cells, an epigenetic switch from H3K27me3 to H3K27ac occurred at specific genomic loci, altering the transcriptional profile. The resulting upregulation of EPHA2 stimulated excessive activation of the prosurvival AKT signaling pathway, leading to radiotherapy resistance. Bromodomain and extraterminal motif (BET) inhibition overcame radiation resistance in H3K27me3-deficient medulloblastoma cells by suppressing H3K27ac levels, blunting EPHA2 overexpression, and mitigating excessive AKT signaling. In addition, BET inhibition sensitized medulloblastoma cells to radiation by enhancing the apoptotic response through suppression of Bcl-xL and upregulation of Bim. This work demonstrates a novel mechanism of radiation resistance in medulloblastoma and identifies an epigenetic marker predictive of radiotherapy response. On the basis of these findings, we propose an epigenetically guided treatment approach targeting radiotherapy resistance in patients with medulloblastoma., Significance: This study demonstrates a novel epigenetic mechanism of radiation resistance in medulloblastoma and identifies a therapeutic approach to improve outcomes in these patients., (©2022 American Association for Cancer Research.)

G9a and EZH2 are two histone methyltransferases commonly upregulated in several cancer types, yet the precise roles that these enzymes play cooperatively in cancer is unclear. We demonstrate here that frequent concurrent upregulation of both G9a and EZH2 occurs in several human tumors. These methyltransferases cooperatively repressed molecular pathways responsible for tumor cell death. In genetically distinct tumor subtypes, concomitant inhibition of G9a and EZH2 potently induced tumor cell death, highlighting the existence of tumor cell survival dependency at the epigenetic level. G9a and EZH2 synergistically repressed expression of genes involved in the induction of endoplasmic reticulum (ER) stress and the production of reactive oxygen species. IL24 was essential for the induction of tumor cell death and was identified as a common target of G9a and EZH2. Loss of function of G9a and EZH2 activated the IL24-ER stress axis and increased apoptosis in cancer cells while not affecting normal cells. These results indicate that G9a and EZH2 promotes the evasion of ER stress-mediated apoptosis by repressing IL24 transcription, therefore suggesting that their inhibition may represent a potential therapeutic strategy for solid cancers., Significance: These findings demonstrate a novel role for G9a and EZH2 histone methyltransferases in suppressing apoptosis, which can be targeted with small molecule inhibitors as a potential approach to improve solid cancer treatment., (©2022 American Association for Cancer Research.)

The methylation of lysine 27 on histone H3 (H3K27me3) is a chromatin mark associated with nucleosome condensation and gene expression silencing. EZH2 is a lysine methyltransferase that catalyzes H3K27me3. In this issue of Cancer Research , Porazzi and colleagues report that pretreatment with EZH2 inhibitors opened up the H3K27me3-marked chromatin of acute myeloid leukemia (AML) cells, which enhanced DNA damage and apoptosis induced by chemotherapeutic agents, in particular the topoisomerase II inhibitors, doxorubicin and etoposide. The EZH2 inhibitor/doxorubicin combination also enabled the expression of proapoptotic genes, potentially contributing to the death of AML cells. This study has significant implications for improving the efficacy of DNA-damaging cytotoxic agents in AML, thereby enabling lower chemotherapy doses and reducing treatment-related side effects. See related article by Porazzi et al., p. 458 ., (©2022 American Association for Cancer Research.)

Bromodomain containing protein 4 (BRD4) plays a critical role in controlling the expression of genes involved in development and cancer. Inactivation of BRD4 inhibits cancer growth, making it a promising anticancer drug target. The cancer stem cell (CSC) population is a key driver of recurrence and metastasis in patients with cancer. Here we show that cancer stem-like cells can be enriched from squamous cell carcinomas (SCC), and that these cells display an aggressive phenotype with enhanced stem cell marker expression, migration, invasion, and tumor growth. BRD4 is highly elevated in this aggressive subpopulation of cells, and its function is critical for these CSC-like properties. Moreover, BRD4 regulates ΔNp63α, a key transcription factor that is essential for epithelial stem cell function that is often overexpressed in cancers. BRD4 regulates an EZH2/STAT3 complex that leads to increased ΔNp63α-mediated transcription. Targeting BRD4 in human SCC reduces ΔNp63α, leading to inhibition of spheroid formation, migration, invasion, and tumor growth. These studies identify a novel BRD4-regulated signaling network in a subpopulation of cancer stem-like cells, elucidating a possible avenue for effective therapeutic intervention. SIGNIFICANCE: This study identifies a signaling cascade driven by BRD4 that upregulates ΔNp63α to promote cancer stem-like properties, which has potential therapeutic implications for the treatment of squamous cell carcinomas., (©2021 American Association for Cancer Research.)

In melanoma metastasis, the role of the AP-2α transcription factor, which is encoded by TFAP2A , is controversial as some findings have suggested tumor suppressor activity while other studies have shown high TFAP2A expression in node-positive melanoma associated with poor prognosis. Here we demonstrate that AP-2α facilitates melanoma metastasis through transcriptional activation of genes within the E2F pathway including EZH2 . A BioID screen found that AP-2α interacts with members of the nucleosome remodeling and deacetylase (NuRD) complex. Loss of AP-2α removed activating chromatin marks in the promoters of EZH2 and other E2F target genes through activation of the NuRD repression complex. In melanoma cells, treatment with tazemetostat, an FDA-approved and highly specific EZH2 inhibitor, substantially reduced anchorage-independent colony formation and demonstrated heritable antimetastatic effects, which were dependent on AP-2α. Single-cell RNA sequencing analysis of a metastatic melanoma mouse model revealed hyperexpansion of Tfap2a High /E2F-activated cell populations in transformed melanoma relative to progenitor melanocyte stem cells. These findings demonstrate that melanoma metastasis is driven by the AP-2α/EZH2 pathway and suggest that AP-2α expression can be used as a biomarker to predict responsiveness to EZH2 inhibitors for the treatment of advanced melanomas. SIGNIFICANCE: AP-2α drives melanoma metastasis by upregulating E2F pathway genes including EZH2 through inhibition of the NuRD repression complex, serving as a biomarker to predict responsiveness to EZH2 inhibitors., (©2021 American Association for Cancer Research.)

Next-generation genomic sequencing has identified multiple novel molecular alterations in cancer. Since the identification of DNA methylation and histone modification, it has become evident that genes encoding epigenetic modifiers that locally and globally regulate gene expression play a crucial role in normal development and cancer progression. The histone methyltransferase enhancer of zeste homolog 2 (EZH2) is the enzymatic catalytic subunit of the polycomb-repressive complex 2 (PRC2) that can alter gene expression by trimethylating lysine 27 on histone 3 (H3K27). EZH2 is involved in global transcriptional repression, mainly targeting tumor-suppressor genes. EZH2 is commonly overexpressed in cancer and shows activating mutations in subtypes of lymphoma. Extensive studies have uncovered an important role for EZH2 in cancer progression and have suggested that it may be a useful therapeutic target. In addition, tumors harboring mutations in other epigenetic genes such as ARID1A, KDM6 , and BAP1 are highly sensitive to EZH2 inhibition, thus increasing its potential as a therapeutic target. Recent studies also suggest that inhibition of EZH2 enhances the response to tumor immunotherapy. Many small-molecule inhibitors have been developed to target EZH2 or the PRC2 complex, with some of these inhibitors now in early clinical trials reporting clinical responses with acceptable tolerability. In this review, we highlight the recent advances in targeting EZH2, its successes, and potential limitations, and we discuss the future directions of this therapeutic subclass., (©2020 American Association for Cancer Research.)

Chemoprevention trials for prostate cancer by androgen receptor or androgen synthesis inhibition have proven ineffective. Recently, it has been demonstrated that the histone methlytransferase, EZH2 is deregulated in mouse and human high-grade prostatic intraepithelial neoplasia (HG-PIN). Using preclinical mouse and human models of prostate cancer, we demonstrate that genetic and chemical disruption of EZH2 expression and catalytic activity reversed the HG-PIN phenotype. Furthermore, inhibition of EZH2 function was associated with loss of cellular proliferation and induction of Tp53-dependent senescence. Together, these data provide provocative evidence for EZH2 as an actionable therapeutic target toward prevention of prostate cancer., (©2020 American Association for Cancer Research.)

T-cell exhaustion in cancer is linked to poor clinical outcomes, where evidence suggests T-cell metabolic changes precede functional exhaustion. Direct competition between tumor-infiltrating lymphocytes (TIL) and cancer cells for metabolic resources often renders T cells dysfunctional. Environmental stress produces epigenome remodeling events within TIL resulting from loss of the histone methyltransferase EZH2. Here, we report an epigenetic mechanism contributing to the development of metabolic exhaustion in TIL. A multiomics approach revealed a Cdkn2a.Arf-mediated, p53-independent mechanism by which EZH2 inhibition leads to mitochondrial dysfunction and the resultant exhaustion. Reprogramming T cells to express a gain-of-function EZH2 mutant resulted in an enhanced ability of T cells to inhibit tumor growth in vitro and in vivo . Our data suggest that manipulation of T-cell EZH2 within the context of cellular therapies may yield lymphocytes that are able to withstand harsh tumor metabolic environments and collateral pharmacologic insults. SIGNIFICANCE: These findings demonstrate that manipulation of T-cell EZH2 in cellular therapies may yield cellular products able to withstand solid tumor metabolic-deficient environments. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/21/4707/F1.large.jpg., (©2020 American Association for Cancer Research.)

Recent studies have thoroughly described genome-wide expression patterns defining molecular subtypes of pancreatic ductal adenocarcinoma (PDAC), with different prognostic and predictive implications. Although the reversible nature of key regulatory transcription circuits defining the two extreme PDAC subtype lineages "classical" and "basal-like" suggests that subtype states are not permanently encoded but underlie a certain degree of plasticity, pharmacologically actionable drivers of PDAC subtype identity remain elusive. Here, we characterized the mechanistic and functional implications of the histone methyltransferase enhancer of zeste homolog 2 (EZH2) in controlling PDAC plasticity, dedifferentiation, and molecular subtype identity. Utilization of transgenic PDAC models and human PDAC samples linked EZH2 activity to PDAC dedifferentiation and tumor progression. Combined RNA- and chromatin immunoprecipitation sequencing studies identified EZH2 as a pivotal suppressor of differentiation programs in PDAC and revealed EZH2-dependent transcriptional repression of the classical subtype defining transcription factor Gata6 as a mechanistic basis for EZH2-dependent PDAC progression. Importantly, genetic or pharmacologic depletion of EZH2 sufficiently increased GATA6 expression, thus inducing a gene signature shift in favor of a less aggressive and more therapy-susceptible, classical PDAC subtype state. Consistently, abrogation of GATA6 expression in EZH2-deficient PDAC cells counteracted the acquisition of classical gene signatures and rescued their invasive capacities, suggesting that GATA6 derepression is critical to overcome PDAC progression in the context of EZH2 inhibition. Together, our findings link the EZH2-GATA6 axis to PDAC subtype identity and uncover EZH2 inhibition as an appealing strategy to induce subtype-switching in favor of a less aggressive PDAC phenotype. SIGNIFICANCE: This study highlights the role of EZH2 in PDAC progression and molecular subtype identity and suggests EZH2 inhibition as a strategy to recalibrate GATA6 expression in favor of a less aggressive disease. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/21/4620/F1.large.jpg., (©2020 American Association for Cancer Research.)

The majority of women diagnosed with epithelial ovarian cancer eventually develop recurrence, which rapidly evolves into chemoresistant disease. Persistence of ovarian cancer stem cells (OCSC) at the end of therapy may be responsible for emergence of resistant tumors. In this study, we demonstrate that in OCSC, the tumor suppressor disabled homolog 2-interacting protein (DAB2IP) is silenced by EZH2-mediated H3K27 trimethylation of the DAB2IP promoter. CRISPR/Cas9-mediated deletion of DAB2IP in epithelial ovarian cancer cell lines upregulated expression of stemness-related genes and induced conversion of non-CSC to CSC, while enforced expression of DAB2IP suppressed CSC properties. Transcriptomic analysis showed that overexpression of DAB2IP in ovarian cancer significantly altered stemness-associated genes and bioinformatic analysis revealed WNT signaling as a dominant pathway mediating the CSC inhibitory effect of DAB2IP. Specifically, DAB2IP inhibited WNT signaling via downregulation of WNT5B, an important stemness inducer. Reverse phase protein array further demonstrated activation of noncanonical WNT signaling via C-JUN as a downstream target of WNT5B, which was blocked by inhibiting RAC1, a prominent regulator of C-JUN activation. Coadministration of EZH2 inhibitor GSK126 and RAC1 inhibitor NSC23766 suppressed OCSC survival in vitro and inhibited tumor growth and increased platinum sensitivity in vivo . Overall, these data establish that DAB2IP suppresses the cancer stem cell phenotype via inhibition of WNT5B-induced activation of C-JUN and can be epigenetically silenced by EZH2 in OCSC. Targeting the EZH2/DAB2IP/C-JUN axis therefore presents a promising strategy to prevent ovarian cancer recurrence and has potential for clinical translation. SIGNIFICANCE: These findings show that combining an epigenetic therapy with a noncanonical WNT signaling pathway inhibitor has the potential to eradicate ovarian cancer stem cells and to prevent ovarian cancer recurrence., (©2020 American Association for Cancer Research.)

Elevated expression of EZH2, the enzymatic subunit of polycomb repressive complex 2 (PRC2), often occurs in cancer. EZH2 expression results in the silencing of genes that suppress tumor formation and metastasis through trimethylation of histone H3 at lysine 27 (H3K27me3) at their promoters. However, inhibitors of EZH2 enzymatic activity have not shown the expected efficacy against cancer in clinical trials, suggesting a need for other strategies to address EZH2 overexpression. Here, we show that SUMOylation, a posttranslational modification characterized by covalent attachment of small ubiquitin-like modifier (SUMO) proteins to a lysine (Lys) residue on target proteins, enhances EZH2 transcription. Either knockdown of the SUMO-activating enzyme SAE2 or pharmacologic inhibition of SUMOylation resulted in decreased levels of EZH2 mRNA and protein as well as reduced H3K27me3 levels. SUMOylation regulated EZH2 expression by enhancing binding of the E2F1 transcriptional activator to the EZH2 promoter. Inhibition of SUMOylation not only resulted in reduced EZH2 mRNA and protein levels but also increased expression of genes silenced by EZH2, such as E-cadherin, which suppresses epithelial-mesenchymal transition and metastasis. In more than 6,500 patient tumor samples across different cancer types, expression of UBA2 and EZH2 was positively correlated. Taken together, our findings suggest that inhibition of SUMOylation may serve as a potential strategy to address EZH2 overexpression and improve current cancer therapeutic approaches. SIGNIFICANCE: These findings provide important biological insights into the mechanism of EZH2 overexpression in cancers and suggest that inhibiting SUMOylation may improve current cancer therapeutic approaches., (©2020 American Association for Cancer Research.)

Tumor progression upon treatment arises from preexisting resistant cancer cells and/or adaptation of persister cancer cells committing to an expansion phase. Here, we show that evasion from viral mimicry response allows the growth of taxane-resistant triple-negative breast cancer (TNBC). This is enabled by an epigenetic state adapted to taxane-induced metabolic stress, where DNA hypomethylation over loci enriched in transposable elements (TE) is compensated by large chromatin domains of H3K27me3 to warrant TE repression. This epigenetic state creates a vulnerability to epigenetic therapy against EZH2, the H3K27me3 methyltransferase, which alleviates TE repression in taxane-resistant TNBC, leading to double-stranded RNA production and growth inhibition through viral mimicry response. Collectively, our results illustrate how epigenetic states over TEs promote cancer progression under treatment and can inform about vulnerabilities to epigenetic therapy. SIGNIFICANCE: Drug-resistant cancer cells represent a major barrier to remission for patients with cancer. Here we show that drug-induced metabolic perturbation and epigenetic states enable evasion from the viral mimicry response induced by chemotherapy in TNBC. These epigenetic states define a vulnerability to epigenetic therapy using EZH2 inhibitors in taxane-resistant TNBC. See related commentary by Janin and Esteller, p. 1258 . This article is highlighted in the In This Issue feature, p. 1241 ., (©2020 American Association for Cancer Research.)

Epigenetic regulation known for DNA methylation and histone modification is critical for securing proper gene expression and chromosomal function, and its aberration induces various pathologic conditions including cancer. Trimethylation of histone H3 on lysine 27 (H3K27me3) is known to suppress various genes related to cancer cell survival and the level of H3K27me3 may have an influence on tumor progression and malignancy. However, it remains unclear how histone methylation is regulated in response to genetic mutation and microenvironmental cues to facilitate the cancer cell survival. Here, we report a novel mechanism of the specific regulation of H3K27me3 by cooperatively two mTOR complexes, mTORC1 and mTORC2 in human glioblastoma (GBM). Integrated analyses revealed that mTORC1 upregulates the protein expression of enhancer of zeste homolog 2, a main component of polycomb repressive complex 2 which is known as H3K27-specific methyltransferase. The other mTOR complex, mTORC2, regulates production of S-adenosylmethionine, an essential substrate for histone methylation. This cooperative regulation causes H3K27 hypermethylation which subsequently promotes tumor cell survival both in vitro and in vivo xenografted mouse tumor model. These results indicate that activated mTORC1 and mTORC2 complexes cooperatively contribute to tumor progression through specific epigenetic regulation, nominating them as an exploitable therapeutic target against cancer. IMPLICATIONS: A dynamic regulation of histone methylation by mTOR complexes promotes tumor growth in human GBM, but at the same time could be exploitable as a novel therapeutic target against this deadly tumor., (©2020 American Association for Cancer Research.)

Aberrant activation of histone methyltransferase EZH2 and ribosome synthesis strongly associate with cancer development and progression. We previously found that EZH2 regulates RNA polymerase III-transcribed 5S ribosomal RNA gene transcription. However, whether EZH2 regulates ribosome synthesis is still unknown. Here, we report that EZH2 promotes ribosome synthesis by targeting and silencing a long noncoding RNA PHACTR2-AS1. PHACTR2-AS1 directly bound ribosome DNA genes and recruited histone methyltransferase SUV39H1, which in turn triggered H3K9 methylation of these genes. Depletion of PHACTR2-AS1 resulted in hyperactivation of ribosome synthesis and instability of ribosomal DNA, which promoted cancer cell proliferation and metastasis. Administration of PHACTR2-AS1-30nt-RNA, which binds to SUV39H1, effectively inhibited breast cancer growth and lung metastasis in mice. PHACTR2-AS1 was downregulated in breast cancer patients, where lower PHACTR2-AS1 expression promoted breast cancer development and correlated with poor patient outcome. Taken together, we demonstrate that PHACTR2-AS1 maintains a H3K9 methylation-marked silent state of ribosomal DNA genes, comprising a regulatory axis that controls breast cancer growth and metastasis. SIGNIFICANCE: These findings reveal that EZH2 mediates ribosomal DNA stability via silencing of PHACTR2-AS1, representing a potential therapeutic target to control breast cancer growth and metastasis., (©2020 American Association for Cancer Research.)

Multiple myeloma remains incurable due to the persistence of a minor population of multiple myeloma cells that exhibit drug resistance, which leads to relapsed and/or refractory multiple myeloma. Elucidating the mechanism underlying drug resistance and developing an effective treatment are critical for clinical management of multiple myeloma. Here we showed that promoting expression of the gene for polycomb-like protein 3 (PHF19) induced multiple myeloma cell growth and multidrug resistance in vitro and in vivo . PHF19 was overexpressed in high-risk and drug-resistant primary cells from patients. High levels of PHF19 were correlated with inferior survival of patients with multiple myeloma, in the Total Therapy 2 cohort and in the Intergroup Francophone du Myeloma (IFM) cohort. Enhancing PHF19 expression levels increased Bcl-xL, Mcl-1 , and HIF-1a expression in multiple myeloma cells. PHF19 also bound directly with EZH2 and promoted the phosphorylation of EZH2 through PDK1/AKT signaling. miR-15a is a small noncoding RNA that targeted the 3'UTR of PHF19 . We found that downregulation of miR-15a led to high levels of PHF19 in multiple myeloma cells. These findings revealed that PHF19 served a crucial role in multiple myeloma proliferation and drug resistance and suggested that the miR-15a/PHF19/EZH2 pathway made a pivotal contribution to multiple myeloma pathogenesis, offering a promising approach to multiple myeloma treatment. IMPLICATIONS: Our findings identify that PHF19 mediates EZH2 phosphorylation as a mechanism of myeloma cell drug resistance, providing a rationale to explore therapeutic potential of targeting PHF19 in relapsed or refractory patients with multiple myeloma., (©2020 American Association for Cancer Research.)

Loss-of-function mutations of EZH2 , the enzymatic component of PRC2, have been associated with poor outcome and chemotherapy resistance in T-cell acute lymphoblastic leukemia (T-ALL). Using isogenic T-ALL cells, with and without CRISPR/Cas9-induced EZH2-inactivating mutations, we performed a cell-based synthetic lethal drug screen. EZH2-deficient cells exhibited increased sensitivity to structurally diverse inhibitors of CHK1, an interaction that could be validated genetically. Furthermore, small-molecule inhibition of CHK1 had efficacy in delaying tumor progression in isogenic EZH2-deficient, but not EZH2 wild-type, T-ALL cells in vivo , as well as in a primary cell model of PRC2-mutant ALL. Mechanistically, EZH2 deficiency resulted in a gene-expression signature of immature T-ALL cells, marked transcriptional upregulation of MYCN , increased replication stress, and enhanced dependency on CHK1 for cell survival. Finally, we demonstrate this phenotype is mediated through derepression of a distal PRC2-regulated MYCN enhancer. In conclusion, we highlight a novel and clinically exploitable pathway in high-risk EZH2-mutated T-ALL. SIGNIFICANCE: Loss-of-function mutations of PRC2 genes are associated with chemotherapy resistance in T-ALL, yet no specific therapy for this aggressive subtype is currently clinically available. Our work demonstrates that loss of EZH2 activity leads to MYCN-driven replication stress, resulting in increased sensitivity to CHK1 inhibition, a finding with immediate clinical relevance. This article is highlighted in the In This Issue feature, p. 890 ., (©2020 American Association for Cancer Research.)

The Hippo pathway regulates cell proliferation and organ size through control of the transcriptional regulators YAP (yes-associated protein) and TAZ. Upon extracellular stimuli such as cell-cell contact, the pathway negatively regulates YAP through cytoplasmic sequestration. Under conditions of low cell density, YAP is nuclear and associates with enhancer regions and gene promoters. YAP is mainly described as a transcriptional activator of genes involved in cell proliferation and survival. Using a genome-wide approach, we show here that, in addition to its known function as a transcriptional activator, YAP functions as a transcriptional repressor by interacting with the multifunctional transcription factor Yin Yang 1 (YY1) and Polycomb repressive complex member enhancer of zeste homologue 2 (EZH2). YAP colocalized with YY1 and EZH2 on the genome to transcriptionally repress a broad network of genes mediating a host of cellular functions, including repression of the cell-cycle kinase inhibitor p27, whose role is to functionally promote contact inhibition. This work unveils a broad and underappreciated aspect of YAP nuclear function as a transcriptional repressor and highlights how loss of contact inhibition in cancer is mediated in part through YAP repressive function. SIGNIFICANCE: This study provides new insights into YAP as a broad transcriptional repressor of key regulators of the cell cycle, in turn influencing contact inhibition and tumorigenesis., (©2020 American Association for Cancer Research.)

Lung metastasis is a major factor affecting long-term survival in patients with adenoid cystic carcinoma. Here, we showed that the long noncoding RNA (lncRNA) MRPL23 antisense RNA 1 (MRPL23-AS1) was highly expressed and correlated with lung metastasis and overall survival in patients with salivary adenoid cystic carcinoma (SACC). MRPL23-AS1 positively regulated epithelial-mesenchymal transition by forming an RNA-protein complex with enhancer of zeste homolog 2 (EZH2). MRPL23-AS1 increased the binding of EZH2 and H3K27me3 on the E-cadherin promoter region. Moreover, MRPL23-AS1 levels were higher in exosomes isolated from the blood plasma of patients with SACC, and exosomal MRPL23-AS1 affected pulmonary microvascular endothelial cells in an "exosomecrine" manner. MRPL23-AS1-enriched exosomes increased microvascular permeability and facilitated the metastasis of SACC in vivo . Collectively, these findings highlight a molecular mechanism of lung metastasis in SACC. MRPL23-AS1 may represent a biomarker and target for clinical intervention to control this intractable disease. SIGNIFICANCE: This study identifies a novel metastasis-promoting lncRNA MRPL23-AS1, which mediates the transcriptional silencing of E-cadherin through forming an RNA-protein complex with EZH2., (©2020 American Association for Cancer Research.)

Mechanisms of intrinsic resistance of serous ovarian cancers to standard treatment with carboplatin and paclitaxel are poorly understood. Seventeen primary serous ovarian cancers classified as responders or nonresponders to standard treatment were screened with DigiWest protein array analysis for 279 analytes. Histone methyl transferase EZH2, an interaction partner of ataxia telangiectasia mutated (ATM), was found as one of the most significantly represented proteins in responsive tumors. Survival analysis of 616 patients confirmed a better outcome in patients with high EZH2 expression, but a worse outcome in patients with low EZH2 and high-ATM-expressing tumors compared with patients with low EZH2 and low-ATM-expressing tumors. A proximity ligation assay further confirmed an association between ATM and EZH2 in tumors of patients with an increased disease-free survival. Knockdown of EZH2 resulted in treatment-resistant cells, but suppression of both EZH2 and ATM, or ATM alone, had no effect. DigiWest protein analysis of EZH2-knockdown cells revealed a decrease in proteins involved in mitotic processes and checkpoint regulation, suggesting that deregulated ATM may induce treatment resistance. IMPLICATIONS: Ovarian cancer is a malignancy with high mortality rates, with to date, no successful molecular characterization strategies. Our study uncovers in a comprehensive approach the involvement of checkpoint regulation via ATM and EZH2, potentially providing a new therapeutic perspective for further investigations., (©2019 American Association for Cancer Research.)

Gliomas are classified by combining histopathologic and molecular features, including isocitrate dehydrogenase ( IDH ) status. Although IDH -wild-type diffuse astrocytic glioma (DAG) shows a more aggressive phenotype than IDH -mutant type, lack of knowledge regarding relevant molecular drivers for this type of tumor has hindered the development of therapeutic agents. Here, we examined human IDH -wild-type DAGs and a glioma mouse model with a mosaic analysis with double markers (MADM) system, which concurrently lacks p53 and NF1 and spontaneously develops tumors highly comparable with human IDH -wild-type DAG without characteristic molecular features of glioblastoma (DAG-nonMF). During tumor formation, enhancer of zeste homolog (EZH2) and the other polycomb repressive complex 2 (PRC2) components were upregulated even at an early stage of tumorigenesis, together with an increased number of genes with H3K27me3 or H3K27me3 and H3K4me3 bivalent modifications. Among the epigenetically dysregulated genes, frizzled-8 ( Fzd8 ), which is known to be a cancer- and stem cell reprogramming-related gene, was gradually silenced during tumorigenesis. Genetic and pharmacologic inhibition of EZH2 in MADM mice showed reactivation of aberrant H3K27me3 target genes, including Fzd8 , together with significant reduction of tumor size. Our study clarifies a pathogenic molecular pathway of IDH -wild-type DAG-nonMF that depends on EZH2 activity and provides a strong rationale for targeting EZH2 as a promising therapeutic approach for this type of glioma. SIGNIFICANCE: EZH2 is involved in the generation of IDH -wild-type diffuse astrocytic gliomas and is a potential therapeutic target for this type of glioma. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/19/4814/F1.large.jpg., (©2019 American Association for Cancer Research.)

Epigenetic gene regulation and metabolism are highly intertwined, yet little is known about whether altered epigenetics influence cellular metabolism during cancer progression. Here, we show that EZH2 and NRAS G12D mutations cooperatively induce progression of myeloproliferative neoplasms to highly penetrant, transplantable, and lethal myeloid leukemias in mice. EZH1, an EZH2 homolog, is indispensable for EZH2-deficient leukemia-initiating cells and constitutes an epigenetic vulnerability. BCAT1, which catalyzes the reversible transamination of branched-chain amino acids (BCAA), is repressed by EZH2 in normal hematopoiesis and aberrantly activated in EZH2-deficient myeloid neoplasms in mice and humans. BCAT1 reactivation cooperates with NRAS G12D to sustain intracellular BCAA pools, resulting in enhanced mTOR signaling in EZH2-deficient leukemia cells. Genetic and pharmacologic inhibition of BCAT1 selectively impairs EZH2-deficient leukemia-initiating cells and constitutes a metabolic vulnerability. Hence, epigenetic alterations rewire intracellular metabolism during leukemic transformation, causing epigenetic and metabolic vulnerabilities in cancer-initiating cells. SIGNIFICANCE: EZH2 inactivation and oncogenic NRAS cooperate to induce leukemic transformation of myeloproliferative neoplasms by activating BCAT1 to enhance BCAA metabolism and mTOR signaling. We uncover a mechanism by which epigenetic alterations rewire metabolism during cancer progression, causing epigenetic and metabolic liabilities in cancer-initiating cells that may be exploited as potential therapeutics. See related commentary by Li and Melnick, p. 1158 . This article is highlighted in the In This Issue feature, p. 1143 ., (©2019 American Association for Cancer Research.)

Granulocytic myeloid infiltration and resultant enhanced neutrophil elastase (NE) activity is associated with poor outcomes in numerous malignancies. We recently showed that NE expression and activity from infiltrating myeloid cells was high in human prostate cancer xenografts and mouse Pten -null prostate tumors. We further demonstrated that NE directly stimulated human prostate cancer cells to proliferate, migrate, and invade, and inhibition of NE in vivo attenuated xenograft growth. Interestingly, reduced expression of SERPINB1, an endogenous NE inhibitor, also correlates with diminished survival in some cancers. Therefore, we sought to characterize the role of SERPINB1 in prostate cancer. We find that SERPINB1 expression is reduced in human metastatic and locally advanced disease and predicts poor outcome. SERPINB1 is also reduced in Pten -null mouse prostate tumors compared with wild-type prostates, and treatment with sivelestat (SERPINB1 pharmacomimetic) attenuates tumor growth. Knockdown of highly expressed SERPINB1 in nonmalignant prostatic epithelial cells (RWPE-1) increases proliferation, decreases apoptosis, and stimulates expression of epithelial-to-mesenchymal transition markers. In contrast, stable SERPINB1 expression in normally low-expressing prostate cancer cells (C4-2) reduces xenograft growth in vivo . Finally, EZH2-mediated histone (H3K27me3) methylation and DNA methyltransferase-mediated DNA methylation suppress SERPINB1 expression in prostate cancer cells. Analysis of The Cancer Genome Atlas and pyrosequencing demonstrate hypermethylation of the SERPINB1 promoter in prostate cancer compared with normal tissue, and the extent of promoter methylation negatively correlates with SERPINB1 mRNA expression. IMPLICATIONS: Our findings suggest that the balance between SERPINB1 and NE is physiologically important within the prostate and may serve as a biomarker and therapeutic target in prostate cancer., (©2019 American Association for Cancer Research.)

Triple-negative breast cancer (TNBC) commonly develops resistance to chemotherapy, yet markers predictive of chemoresistance in this disease are lacking. Here, we define WNT10B-dependent biomarkers for β-CATENIN/HMGA2/EZH2 signaling predictive of reduced relapse-free survival. Concordant expression of HMGA2 and EZH2 proteins is observed in MMTV - Wnt10b LacZ transgenic mice during metastasis, and Hmga2 haploinsufficiency decreased EZH2 protein expression, repressing lung metastasis. A novel autoregulatory loop interdependent on HMGA2 and EZH2 expression is essential for β-CATENIN/TCF-4/LEF-1 transcription. Mechanistically, both HMGA2 and EZH2 displaced Groucho/TLE1 from TCF-4 and served as gatekeepers for K49 acetylation on β-CATENIN, which is essential for transcription. In addition, we discovered that HMGA2-EZH2 interacts with the PRC2 complex. Absence of HMGA2 or EZH2 expression or chemical inhibition of Wnt signaling in a chemoresistant patient-derived xenograft (PDX) model of TNBC abolished visceral metastasis, repressing AXIN2, MYC, EZH2, and HMGA2 expression i n vivo . Combinatorial therapy of a WNT inhibitor with doxorubicin synergistically activated apoptosis in vitro , resensitized PDX-derived cells to doxorubicin, and repressed lung metastasis in vivo . We propose that targeting the WNT10B biomarker network will provide improved outcomes for TNBC. SIGNIFICANCE: These findings reveal targeting the WNT signaling pathway as a potential therapeutic strategy in triple-negative breast cancer. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/5/982/F1.large.jpg., (©2018 American Association for Cancer Research.)

Aberrant activation of β-catenin signaling is a critical driver for tumorigenesis, but the mechanism underlying this activation is not completely understood. In this study, we demonstrate a critical role of β-catenin signaling in stabilization of enhancer of zeste homolog 2 (EZH2) and control of EZH2-mediated gene repression in oncogenesis. β-Catenin/TCF4 activated the transcription of the deubiquitinase USP1, which then interacted with and deubiquitinated EZH2 directly. USP1-mediated stabilization of EZH2 promoted its recruitment to the promoters of CDKN1B, RUNX3, and HOXA5, resulting in enhanced enrichment of histone H3K27me3 and repression of target gene expression. In human glioma specimens, expression levels of nuclear β-catenin, USP1, and EZH2 correlated with one another. Depletion of β-catenin/USP1/EZH2 repressed glioma cell proliferation in vitro and tumor formation in vivo . Our findings indicate that a β-catenin-USP1-EZH2 axis orchestrates the interplay between dysregulated β-catenin signaling and EZH2-mediated gene epigenetic silencing during glioma tumorigenesis. SIGNIFICANCE: These findings identify the β-catenin-USP1-EZH2 signaling axis as a critical mechanism for glioma tumorigenesis that may serve as a new therapeutic target in glioblastoma., (©2018 American Association for Cancer Research.)

Advanced prostate cancer initially responds to androgen deprivation therapy (ADT), but the disease inevitably recurs as castration-resistant prostate cancer (CRPC). Although CRPC initially responds to abiraterone and enzalutamide, the disease invariably becomes nonresponsive to these agents. Novel approaches are required to circumvent resistance pathways and to extend survival, but the mechanisms underlying resistance remain poorly defined. Our group previously showed the histone lysine- N -methyltransferase EZH2 to be overexpressed in prostate cancer and quantitatively associated with progression and poor prognosis. In this study, we screened a library of epigenetic inhibitors for their ability to render CRPC cells sensitive to enzalutamide and found that EZH2 inhibitors specifically potentiated enzalutamide-mediated inhibition of proliferation. Moreover, we identified antisense oligonucleotides (ASO) as a novel drug strategy to ablate EZH2 and androgen receptor (AR) expression, which may have advantageous properties in certain settings. RNA-seq, chromatin immunoprecipitation sequencing, and assay for transposase-accessible chromatin using sequencing demonstrated that EZH2 inhibition altered the AR cistrome to significantly upregulate AR signaling, suggesting an enhanced dependence of CRPC cells on this pathway following inhibition of EZH2. Combination treatment with ASO targeting EZH2 and AR transcripts inhibited prostate cancer cell growth in vitro and in vivo better than single agents. In sum, this study identifies EZH2 as a critical epigenetic regulator of ADT resistance and defines ASO-based cotargeting of EZH2 and AR as a promising strategy for the treatment of CRPC. Significance: Simultaneous targeting of lysine methyltransferase EZH2 and the AR with ASO proves a novel and effective therapeutic strategy in patients with CRPC. Cancer Res; 78(20); 5731-40. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Resistance to cancer treatment can be driven by epigenetic reprogramming of specific transcriptomes in favor of the refractory phenotypes. Here we discover that tamoxifen resistance in breast cancer is driven by a regulatory axis consisting of a master transcription factor, its cofactor, and an epigenetic regulator. The oncogenic histone methyltransferase EZH2 conferred tamoxifen resistance by silencing the expression of the estrogen receptor α (ERα) cofactor GREB1. In clinical specimens, induction of DNA methylation of a particular CpG-enriched region at the GREB1 promoter negatively correlated with GREB1 levels and cell sensitivity to endocrine agents. GREB1 also ensured proper cellular reactions to different ligands by recruiting distinct sets of ERα cofactors to cis -regulatory elements, which explains the contradictory biological effects of GREB1 on breast cancer cell growth in response to estrogen or antiestrogen. In refractory cells, EZH2-dependent repression of GREB1 triggered chromatin reallocation of ERα coregulators, converting the antiestrogen into an agonist. In clinical specimens from patients receiving adjuvant tamoxifen treatment, expression levels of EZH2 and GREB1 were correlated negatively, and taken together better predicted patient responses to endocrine therapy. Overall, our work suggests a new strategy to overcome endocrine resistance in metastatic breast cancer by targeting a particular epigenetic program. Significance: This study suggests a new strategy to overcome endocrine resistance in metastatic breast cancer by targeting a particular epigenetic program defined within. Cancer Res; 78(3); 671-84. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Acquired and intrinsic resistance to receptor tyrosine kinase inhibitors (RTKi) represents a major hurdle in improving the management of clear cell renal cell carcinoma (ccRCC). Recent reports suggest that drug resistance is driven by tumor adaptation via epigenetic mechanisms that activate alternative survival pathways. The histone methyl transferase EZH2 is frequently altered in many cancers, including ccRCC. To evaluate its role in ccRCC resistance to RTKi, we established and characterized a spontaneously metastatic, patient-derived xenograft model that is intrinsically resistant to the RTKi sunitinib, but not to the VEGF therapeutic antibody bevacizumab. Sunitinib maintained its antiangiogenic and antimetastatic activity but lost its direct antitumor effects due to kinome reprogramming, which resulted in suppression of proapoptotic and cell-cycle-regulatory target genes. Modulating EZH2 expression or activity suppressed phosphorylation of certain RTKs, restoring the antitumor effects of sunitinib in models of acquired or intrinsically resistant ccRCC. Overall, our results highlight EZH2 as a rational target for therapeutic intervention in sunitinib-resistant ccRCC as well as a predictive marker for RTKi response in this disease. Cancer Res; 77(23); 6651-66. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Deregulation of the Wnt/β-catenin signaling pathway drives the development of colorectal cancer, but understanding of this pathway remains incomplete. Here, we report that the damage-specific DNA-binding protein DDB2 is critical for β-catenin-mediated activation of RNF43, which restricts Wnt signaling by removing Wnt receptors from the cell surface. Reduced expression of DDB2 and RNF43 was observed in human hyperplastic colonic foci. DDB2 recruited EZH2 and β-catenin at an upstream site in the Rnf43 gene, enabling functional interaction with distant TCF4/β-catenin-binding sites in the intron of Rnf43 This novel activity of DDB2 was required for RNF43 function as a negative feedback regulator of Wnt signaling. Mice genetically deficient in DDB2 exhibited increased susceptibility to colon tumor development in a manner associated with higher abundance of the Wnt receptor-expressing cells and greater activation of the downstream Wnt pathway. Our results identify DDB2 as both a partner and regulator of Wnt signaling, with an important role in suppressing colon cancer development. Cancer Res; 77(23); 6562-75. ©2017 AACR ., (©2017 American Association for Cancer Research.)

DNA demethylases of the TET family function as tumor suppressors in various human cancers, but their pathogenic contributions and mechanisms of action in gastric carcinogenesis and progression remain unclear. Here, we report that TET is transcriptionally upregulated in gastric cancer, where it correlates with poor prognosis. Mechanistic investigations revealed that TET facilitated gastric carcinogenesis through a noncoding function of the 3'UTR, which interacted with miR-26. This interaction resulted in sequestration of miR-26 from its target EZH2, which released the suppression on EZH2, and thereby led to EZH2 overexpression in gastric cancer. Our findings uncover a novel noncoding function for TET family proteins in facilitating gastric carcinogenesis. Cancer Res; 77(22); 6069-82. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Gliomas with mutant p53 occurring in 30% of glioma patients exhibit therapeutic resistance and poor outcomes. In this study, we identify a novel mechanism through which mutant p53 drives cancer cell survival and malignant growth. We documented overexpression of the zinc finger protein ZDHHC5 in glioma compared with normal brain tissue and that this event tightly correlated with p53 mutations. Mechanistic investigations revealed that mutant p53 transcriptionally upregulated ZDHHC5 along with the nuclear transcription factor NF-Y. These events contributed to the development of glioma by promoting the self-renewal capacity and tumorigenicity of glioma stem-like cells, by altering the palmitoylation and phosphorylation status of the tumor suppressor EZH2. Taken together, our work highlighted ZDHHC5 as a candidate therapeutic target for management of p53-mutated gliomas. Cancer Res; 77(18); 4998-5010. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Medulloblastoma is the most common malignant brain tumor in children. Although accumulated research has suggested that cancer stem-like cells play a key role in medulloblastoma tumorigenesis, the specific molecular mechanism regarding proliferation remains elusive. Here, we reported more abundant expression of maternal embryonic leucine-zipper kinase (MELK) and enhancer of zeste homolog 2 (EZH2) in medulloblastoma stem-like cells than in neural stem cells and the interaction between the two proteins could mediate the self-renewal of sonic hedgehog subtype medulloblastoma. In human medulloblastoma, extensive nodularity and large-cell/anaplastic subgroups differed according to the staining levels of MELK and EZH2 from the other two subgroups. The proportion of MELK- or EZH2-positive staining status could be considered as a potential indicator for survival. Mechanistically, MELK bound to and phosphorylated EZH2, and its methylation was induced by EZH2 in medulloblastoma, which could regulate the proliferation of cancer stem-like cells. In xenografts, loss of MELK or EZH2 attenuated medulloblastoma stem-like cell-derived tumor growth and promoted differentiation. These findings indicate that MELK-induced phosphorylation and EZH2-mediated methylation in MELK/EZH2 pathway are essential for medulloblastoma stem-like cell-derived tumor proliferation, thereby identifying a potential therapeutic strategy for these patients. Implications: This study demonstrates that the interaction occurring between MELK and EZH2 promotes self-proliferation and stemness, thus representing an attractive therapeutic target and potential candidate for diagnosis of medulloblastoma. Mol Cancer Res; 15(9); 1275-86. ©2017 AACR ., (©2017 American Association for Cancer Research.)

In multiple myeloma, osteolytic lesions rarely heal because of persistent suppressed osteoblast differentiation resulting in a high fracture risk. Herein, chromatin immunoprecipitation analyses reveal that multiple myeloma cells induce repressive epigenetic histone changes at the Runx2 locus that prevent osteoblast differentiation. The most pronounced multiple myeloma-induced changes were at the Runx2-P1 promoter, converting it from a poised bivalent state to a repressed state. Previously, it was observed that multiple myeloma induces the transcription repressor GFI1 in osteoblast precursors, which correlates with decreased Runx2 expression, thus prompting detailed characterization of the multiple myeloma and TNFα-dependent GFI1 response element within the Runx2-P1 promoter. Further analyses reveal that multiple myeloma-induced GFI1 binding to Runx2 in osteoblast precursors and recruitment of the histone modifiers HDAC1, LSD1, and EZH2 is required to establish and maintain Runx2 repression in osteogenic conditions. These GFI1-mediated repressive chromatin changes persist even after removal of multiple myeloma. Ectopic GFI1 is sufficient to bind to Runx2 , recruit HDAC1 and EZH2, increase H3K27me3 on the gene, and prevent osteogenic induction of endogenous Runx2 expression. Gfi1 knockdown in MC4 cells blocked multiple myeloma-induced recruitment of HDAC1 and EZH2 to Runx2 , acquisition of repressive chromatin architecture, and suppression of osteoblast differentiation. Importantly, inhibition of EZH2 or HDAC1 activity in pre-osteoblasts after multiple myeloma exposure in vitro or in osteoblast precursors from patients with multiple myeloma reversed the repressive chromatin architecture at Runx2 and rescued osteoblast differentiation. Implications: This study suggests that therapeutically targeting EZH2 or HDAC1 activity may reverse the profound multiple myeloma-induced osteoblast suppression and allow repair of the lytic lesions. Mol Cancer Res; 15(4); 405-17. ©2017 AACR ., (©2017 American Association for Cancer Research.)

The lethal phenotype of castration-resistant prostate cancer (CRPC) is generally caused by augmented signaling from the androgen receptor (AR). Here, we report that the AR-repressed gene CCN3/NOV inhibits AR signaling and acts in a negative feedback loop to block AR function. Mechanistically, a cytoplasmic form of CCN3 interacted with the AR N-terminal domain to sequester AR in the cytoplasm of prostate cancer cells, thereby reducing AR transcriptional activity and inhibiting cell growth. However, constitutive repression of CCN3 by the Polycomb group protein EZH2 disrupted this negative feedback loop in both CRPC and enzalutamide-resistant prostate cancer cells. Notably, restoring CCN3 was sufficient to effectively reduce CPRC cell proliferation in vitro and to abolish xenograft tumor growth in vivo Taken together, our findings establish CCN3 as a pivotal regulator of AR signaling and prostate cancer progression and suggest a functional intersection between Polycomb and AR signaling in CRPC. Cancer Res; 77(2); 412-22. ©2016 AACR., Competing Interests: of Potential Conflicts of Interest No potential conflicts of interest were disclosed., (©2016 American Association for Cancer Research.)

Long noncoding RNAs (lncRNA) have been implicated in human cancer but their mechanisms of action are mainly undocumented. In this study, we investigated lncRNA alterations that contribute to gastric cancer through an analysis of The Cancer Genome Atlas RNA sequencing data and other publicly available microarray data. Here we report the gastric cancer-associated lncRNA HOXA11-AS as a key regulator of gastric cancer development and progression. Patients with high HOXA11-AS expression had a shorter survival and poorer prognosis. In vitro and in vivo assays of HOXA11-AS alterations revealed a complex integrated phenotype affecting cell growth, migration, invasion, and apoptosis. Strikingly, high-throughput sequencing analysis after HOXA11-AS silencing highlighted alterations in cell proliferation and cell-cell adhesion pathways. Mechanistically, EZH2 along with the histone demethylase LSD1 or DNMT1 were recruited by HOXA11-AS, which functioned as a scaffold. HOXA11-AS also functioned as a molecular sponge for miR-1297, antagonizing its ability to repress EZH2 protein translation. In addition, we found that E2F1 was involved in HOXA11-AS activation in gastric cancer cells. Taken together, our findings support a model in which the EZH2/HOXA11-AS/LSD1 complex and HOXA11-AS/miR-1297/EZH2 cross-talk serve as critical effectors in gastric cancer tumorigenesis and progression, suggesting new therapeutic directions in gastric cancer. Cancer Res; 76(21); 6299-310. ©2016 AACR., (©2016 American Association for Cancer Research.)

Unlabelled: As a master regulator of chromatin function, the lysine methyltransferase EZH2 orchestrates transcriptional silencing of developmental gene networks. Overexpression of EZH2 is commonly observed in human epithelial cancers, such as non-small cell lung carcinoma (NSCLC), yet definitive demonstration of malignant transformation by deregulated EZH2 remains elusive. Here, we demonstrate the causal role of EZH2 overexpression in NSCLC with new genetically engineered mouse models of lung adenocarcinoma. Deregulated EZH2 silences normal developmental pathways, leading to epigenetic transformation independent of canonical growth factor pathway activation. As such, tumors feature a transcriptional program distinct from KRAS- and EGFR-mutant mouse lung cancers, but shared with human lung adenocarcinomas exhibiting high EZH2 expression. To target EZH2-dependent cancers, we developed a potent open-source EZH2 inhibitor, JQEZ5, that promoted the regression of EZH2-driven tumors in vivo, confirming oncogenic addiction to EZH2 in established tumors and providing the rationale for epigenetic therapy in a subset of lung cancer., Significance: EZH2 overexpression induces murine lung cancers that are similar to human NSCLC with high EZH2 expression and low levels of phosphorylated AKT and ERK, implicating biomarkers for EZH2 inhibitor sensitivity. Our EZH2 inhibitor, JQEZ5, promotes regression of these tumors, revealing a potential role for anti-EZH2 therapy in lung cancer. Cancer Discov; 6(9); 1006-21. ©2016 AACR.See related commentary by Frankel et al., p. 949This article is highlighted in the In This Issue feature, p. 932., Competing Interests: P.K.R. is a consultant to Selecta Biosciences Inc, Watertown, MA. J.E.B. is a Scientific Founder of SHAPE Pharmaceuticals, Acetylon Pharmaceuticals, Tensha Therapeutics and C4 Therapeutics and is the inventor on IP licensed to these entities. J.E.B. currently an employee of Novartis Institutes of Biomedical Research., (©2016 American Association for Cancer Research.)