EZH2 Methyltransferase Activity Research Articles

The chromatin remodeler DEK promotes proliferation of mammary epithelium and is associated with H3K27me3 epigenetic modifications.

The DEK chromatin remodeling protein was previously shown to confer oncogenic phenotypes to human and mouse mammary epithelial cells using in vitro and knockout mouse models. However, its functional role in normal mammary gland epithelium remained unexplored. We developed two novel mouse models to study the role of Dek in normal mammary gland biology in vivo . Mammary gland-specific Dek over-expression in mice resulted in hyperproliferation of cells that visually resembled alveolar cells, and a transcriptional profile that indicated increased expression of cell cycle, mammary stem/progenitor, and lactation-associated genes. Conversely, Dek knockout mice exhibited an alveologenesis or lactation defect, resulting in dramatically reduced pup survival. Analysis of previously published single-cell RNA-sequencing of mouse mammary glands revealed that Dek is most highly expressed in mammary stem cells and alveolar progenitor cells, and to a lesser extent in basal epithelial cells, supporting the observed phenotypes. Mechanistically, we discovered that Dek is a modifier of Ezh2 methyltransferase activity, upregulating the levels of histone H3 trimethylation on lysine 27 (H3K27me3) to control gene transcription. Combined, this work indicates that Dek promotes proliferation of mammary epithelial cells via cell cycle deregulation. Furthermore, we report a novel function for Dek in alveologenesis and histone H3 K27 trimethylation.

EZH2 represses mesenchymal genes and upholds the epithelial state of breast carcinoma cells

Emerging studies support that the polycomb repressive complex 2 (PRC2) regulates phenotypic changes of carcinoma cells by modulating their shifts among metastable states within the epithelial and mesenchymal spectrum. This new role of PRC2 in cancer has been recently proposed to stem from the ability of its catalytic subunit EZH2 to bind and modulate the transcription of mesenchymal genes during epithelial-mesenchymal transition (EMT) in lung cancer cells. Here, we asked whether this mechanism is conserved in other types of carcinomas. By combining TGF-β-mediated reversible induction of epithelial to mesenchymal transition and inhibition of EZH2 methyltransferase activity, we demonstrate that EZH2 represses a large set of mesenchymal genes and favours the residence of breast cancer cells towards the more epithelial spectrum during EMT. In agreement, analysis of human patient samples supports that EZH2 is required to efficiently repress mesenchymal genes in breast cancer tumours. Our results indicate that PRC2 operates through similar mechanisms in breast and lung cancer cells. We propose that PRC2-mediated direct transcriptional modulation of the mesenchymal gene expression programme is a conserved molecular mechanism underlying cell dissemination across human carcinomas.

Abstract PO1-24-05: EZH2 histone methyltransferase activity promotes spindle metaplastic breast carcinoma metastasis and is induced by CCN6 knockout/b-catenin/TCF axis

Abstract Background: Metaplastic breast carcinomas (mBrCAs) are a highly aggressive subtype of triple negative breast cancer with histological evidence of deregulated differentiation towards non-glandular components. Previous studies have demonstrated that human mBrCA often exhibit activation of differentiation pathways, including canonical Wnt/b-catenin and EZH2-mediated transcriptional repression. Our lab has identified CCN6 as a tumor suppressor in mBrCA. MMTV-Cre;Ccn6fl/fl (CCN6KO) mice develop spindle mBrCAs and CCN6 is reduced/lost in 68% of human mBrCAs. Recently, we have demonstrated that the tumor suppressor function of CCN6 in spindle mBrCA requires activation of Wnt/b-catenin signaling pathway. Here, we tested the hypothesis that CCN6 KO leads to the upregulation of EZH2 histone methyltransferase promoting mBrCA. Furthermore, we investigated the requirement for the activation of the Wnt/b-catenin pathway in this mechanism. Methods: To test the effect of CCN6/b-catenin on EZH2 gene and protein expression we performed IHC, IF, qRT-PCR, ChIP-Seq, RNA-seq, invasion and adhesion assays, EZH2 reporter assay and immunoblots in mBrCA cell lines and MMTV-Cre;Ccn6fl/fl tumors. To investigate the role of CCN6KO-induced b-catenin activation on EZH2 activity and neoplastic functions we employed three independent approaches: i) Expression of a dominant-negative Tcf4 (dnTcf4) rescued with EZH2-WT, dSET and dNLS domain mutants versus vector in MMTV-Cre;Ccn6fl/fl tumor-derived cells; ii) Expression of a constitutively active mutant (S33Y) b-catenin in concert with treatment with recombinant human CCN6 (rhCCN6; 500 ug/ml) versus control; iii) Syngeneic orthotopic mammary tumor transplants of MMTV-Cre;Ccn6fl/fl were used for in vivo rescue experiments with rhCCN6 or BSA. To assess the therapeutic benefit of inhibiting EZH2 methyltransferase activity, MMTV-Cre;Ccn6fl/fl tumor cells (CCN6KO cells) were implanted orthotopically or intracardially in FVB mice, followed by treatment with EPZ-6438 (a selective EZH2 methyltransferase inhibitor) or vehicle. We tested CCN6, b-catenin, and EZH2 expression by IHC in a cohort of 27 human mBrCA tumor samples. Results: CCN6KO-induced b-catenin/TCF activation mediates EZH2 transcriptional upregulation and the deposition of repressive H3K27me3 in spindle mBrCAs. We found that the invasion program triggered by CCN6KO-induced Wnt/b-catenin requires EZH2 catalytic activity as WT-EZH2 (but not dSET-EZH2) rescued the reduced invasion of CCN6KO-dnTcf4 cells. RNA-seq and 3H3K27 ChIP-seq of CCN6KO-dnTcf4 cells transduced with EZH2-WT or dSET-EZH2 identify specific CCN6KO-b-catenin/Tcf targets that require EZH2 transcriptional repressor function. In vivo, administration of CCN6 protein to MMTV-Cre;Ccn6fl/fl tumor transplants reduces tumor growth and nuclear b-catenin, EZH2 and 3H3K27 in the tumors. Pharmacologic inhibition of EZH2 reduces the growth and metastasis of CCN6KO mBrCA tumors and improves survival. We identify a subset of human spindle mBrCA (54%) that display a CCN6Low/nuclear b-cat/EZH2High phenotype. Conclusion: We found a critical role for EZH2 activation in CCN6-deficient mBrCA tumor phenotypes via b-catenin/TCF Wnt canonical signaling. We demonstrate the effectiveness of pharmacological inhibition of EZH2 methyltransferase activity in reducing primary tumor growth and distant metastasis in mouse models of spindle mBrCA. In clinical samples, low CCN6 is significantly associated with activated b-catenin and high EZH2 in spindle mBrCAs compared to other subtypes. These data reveal a novel tumor suppressor mechanism of CCN6 and provide compelling evidence supporting the potential therapeutic value of CCN6 restoration, b-catenin or EZH2 inhibition as promising approaches for the treatment of spindle mBrCAs. Citation Format: Maria Gonzalez, Ahmad Eido, GIUSEPPINA AUGIMERI, Celina Kleer. EZH2 histone methyltransferase activity promotes spindle metaplastic breast carcinoma metastasis and is induced by CCN6 knockout/b-catenin/TCF axis [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-24-05.

Abstract 1727: Cytoplasmic EZH2 and the DLC1 tumor suppressor are regulated by the CRM1 nuclear export protein and by specific complex formation between mutant KRAS and RAN-GAP

Abstract The DLC1 tumor suppressor gene encodes a cytoplasmic RHO-GAP protein whose expression is frequently decreased in many cancer types, especially lung cancer. We have identified several cancer-associated post-translational modifications in the DLC1 protein that decrease its tumor suppressor activity and are exploring the upstream mechanisms that regulate the changes as well as the therapeutic potential of reactivating the tumor suppressor activity of DLC1 by inhibiting the post-translational changes. We have made the unexpected observation that lung cancer expresses increased levels of EZH2 in the cytoplasm, where it can methylate DLC1, which can trigger the ubiquitin-proteasomal-dependent degradation of DLC1. The cytoplasmic EZH2 is derived from the nucleus, and inhibition of the CRM1 nuclear export protein or the EZH2 methyl transferase activity can drastically reduce the level of cytoplasmic EZH2 or its activity, which increases the half-life and steady-state level of DLC1 protein several fold. The stabilized DLC1 protein can be reactivated by inhibiting the AKT and SRC kinases, whose phosphorylation of DLC1 can attenuate its tumor suppressor function. A three-drug combination of an EZH2 or a CRM1 inhibitor, together with AKT and SRC kinase inhibitors, can greatly reduce the weight of tumor xenografts from human lung cancer cell lines and induce markers of apoptosis and senescence. Remarkably, isogenic knock-out of DLC1 abolishes most of the anti-tumor activity, implying that DLC1 is the main target of the three-drug treatment, although each inhibitor has many other targets. In addition, we have found that inhibiting endogenous mutant KRAS in the lung cancer lines can phenocopy the CRM1 inhibitor, leading to loss of cytoplasmic EZH2 and increasing the level of DLC1 protein. Conversely, mutant KRAS increases the level of cytoplasmic EZH2 and decreases DLC1. We have investigated the underlying mechanism, and have determined that mutant KRAS regulates nucleocytoplasmic protein shuttling by forming a previously unidentified complex with RAN-GAP (present in primary human tumors and cell lines), which regulates the RAN GTPase, a protein that is required for other proteins to shuttle from the cytoplasm to the nucleus and from the nucleus to the cytoplasm. Formation of the RAS/RAN-GAP complex and its downstream consequences are independent of known RAS signaling from the plasma membrane, as inhibition of MAPK and PI3K does not affect this process. However, efficient complex formation does depend on farnesylation of RAS, as farnesylation-deficient mutants form the complex less efficiently and have reduced consequences for EZH2 and DLC1. Thus, we have identified nucleocytoplasmic protein shuttling as a new function for mutant KRAS and have determined the mechanism depends on complex formation between KRAS and RAN-GAP. Citation Format: Brajendra Tripathi, Nicole Hirsh, David Hansen, Xiaolan Qian, Dunrui Wang, Marian E. Durkin, Ashley Lim, Alex G. Papageorge, Suresh S. Ramalingham, Adam I. Marcus, Douglas R. Lowy. Cytoplasmic EZH2 and the DLC1 tumor suppressor are regulated by the CRM1 nuclear export protein and by specific complex formation between mutant KRAS and RAN-GAP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1727.

Pharmacological inhibition of EZH2 by ZLD1039 suppresses tumor growth and pulmonary metastasis in melanoma cells in vitro and in vivo

The incidence and mortality rate of malignant melanoma are increasing worldwide. Metastasis reduces the efficacy of current melanoma therapies and leads to poor prognosis for patients. EZH2 is a methyltransferase that promotes the proliferation, metastasis, and drug resistance of tumor cells by regulating transcriptional activity. EZH2 inhibitors could be effective in melanoma therapies. Herein, we aimed to investigate whether the pharmacological inhibition of EZH2 by ZLD1039, a potent and selective S-adenosyl-l-methionine-EZH2 inhibitor, suppresses tumor growth and pulmonary metastasis in melanoma cells. Results showed that ZLD1039 selectively reduced H3K27 methylation in melanoma cells by inhibiting EZH2 methyltransferase activity. Additionally, ZLD1039 exerted excellent antiproliferative effects on melanoma cells in 2D and 3D culture systems. Administration of ZLD1039 (100 mg/kg) by oral gavage caused antitumor effects in the A375 subcutaneous xenograft mouse model. RNA sequencing and GSEA revealed that the ZLD1039-treated tumors exhibited changes in the gene sets enriched from the “Cell Cycle” and “Oxidative Phosphorylation”, whereas the “ECM receptor interaction” gene set had a negative enrichment score. Mechanistically, ZLD1039 induced G0/G1 phase arrest by upregulating p16 and p27 and inhibiting the functions of the cyclin D1/CDK6 and cyclin E/CDK2 complexes. Moreover, ZLD1039 induced apoptosis in melanoma cells via the mitochondrial reactive oxygen species apoptotic pathway, consistent with the changes in transcriptional signatures. ZLD1039 also exhibited excellent antimetastatic effects on melanoma cells in vitro and in vivo. Our data highlight that ZLD1039 may be effective against melanoma growth and pulmonary metastasis and thus could serve as a therapeutic agent for melanoma.

Inhibition of pancreatic EZH2 restores progenitor insulin in T1D donor

Type 1 diabetes (T1D) is an autoimmune disease that selectively destroys insulin-producing β-cells in the pancreas. An unmet need in diabetes management, current therapy is focussed on transplantation. While the reprogramming of progenitor cells into functional insulin-producing β-cells has also been proposed this remains controversial and poorly understood. The challenge is determining why default transcriptional suppression is refractory to exocrine reactivation. After the death of a 13-year-old girl with established insulin-dependent T1D, pancreatic cells were harvested in an effort to restore and understand exocrine competence. The pancreas showed classic silencing of β-cell progenitor genes with barely detectable insulin (Ins) transcript. GSK126, a highly selective inhibitor of EZH2 methyltransferase activity influenced H3K27me3 chromatin content and transcriptional control resulting in the expression of core β-cell markers and ductal progenitor genes. GSK126 also reinstated Ins gene expression despite absolute β-cell destruction. These studies show the refractory nature of chromatin characterises exocrine suppression influencing β-cell plasticity. Additional regeneration studies are warranted to determine if the approach of this n-of-1 study generalises to a broader T1D population.

Abstract SY24-03: Modulating cellular senescence to reinstate natural killer cell immunity for pancreatic cancer immunotherapy

Abstract Background: Cancer immunotherapies can lead to durable and sometimes curative responses in patients with otherwise treatment-refractory disease, and as such have revolutionized our approach to cancer therapy. Still, there are many roadblocks that must be overcome to make immunotherapies effective across a broader spectrum of patients and cancer types. Immune checkpoint blockade (ICB) therapies that are effective in some malignancies through reactivation of cytotoxic T cell immunity have failed in immunologically “cold” tumors such as pancreatic ductal adenocarcinoma (PDAC) due to poor T cell infiltration and weak antigen presentation necessary for effective anti-tumor T cell responses. We and others have demonstrated that induction of cellular senescence and the senescence associated secretory phenotype (SASP), which leads to production of immunomodulatory cytokines, chemokines, and growth factors, can be a powerful way to not only enhance T cell infiltration into tumors but also reactivate a different type of innate, antigen-independent Natural Killer (NK) cell immunity that can mediate tumor control. Recently, we found that a combination of two RAS pathway targeting therapies- the MEK inhibitor trametinib and CDK4/6 inhibitor palbociclib (T/P)- could induce senescence in genetically engineered mouse models of KRAS mutant lung adenocarcinoma and PDAC (Ruscetti*, Leibold*, Bott* et al. Science 2018; Ruscetti*, Morris*, Mezzadra* et al. Cell 2020). Strikingly, whereas therapy-induced senescence led to NK cell-mediated tumor regressions and long-term survival benefit in KRAS mutant lung cancer, it had little effect on NK cell immunity and tumor progression in PDAC. Here we set out to understand how the pancreas tumor microenvironment (TME) suppresses NK immunity and develop strategies to harness NK cell surveillance for immunotherapy in PDAC. Results and Discussion: We took advantage of genetically similar Kras mutant, p53 altered PDAC and lung cancer cell lines that could be transplanted into different organs of C57BL/6 mice to study whether and how the pancreas TME may impact senescence-driven immune responses. Syngeneic KRAS mutant lung and PDAC tumor cells transplanted into the pancreas of C57BL/6 mice displayed transcriptional repression and reduced chromatin accessibility of SASP transcriptional activators (NF-KB, IRFs) and factors necessary for NK cell activity (IL-15, IL-18) and chemotaxis (CCL2, CCL7, CCL8, CXCL9, CXCL10) following T/P-induced senescence, and did not undergo anti-tumor NK immune surveillance as compared to tumors propagated in the lungs and liver. Transcriptomic analysis identified induction of EZH2 and epigenetic repression of its targets, including key SASP factors and regulators, specifically in tumor cells grown in the pancreas TME. Genetic or pharmacological inhibition of EZH2 enhanced proinflammatory SASP signaling and resulted in NK cell migration and anti-tumor cytotoxicity in murine and human PDAC lines and transplanted and genetically engineered PDAC mouse models. Remarkably, tumor-intrinsic EZH2 knockdown in combination with T/P treatment led to complete tumor regressions in some PDAC-bearing mice that were reversed following NK cell depletion or suppression of key SASP factors. Pharmacological inhibition of EZH2 methyltransferase activity also enhanced NK cell infiltration and tumor control following therapy-induced senescence in transplanted and genetically engineered PDAC mouse models. Importantly, we found that an EZH2 repression signature was associated with enhanced NK cell infiltration and SASP induction in human PDAC patient samples. These results demonstrate that EZH2 mediates transcriptional repression of the proinflammatory SASP in the pancreas TME, and that EZH2 blockade in combination with senescence-inducing therapies could be a powerful means to reactivate absent NK cell surveillance in PDAC to achieve immune-mediated tumor responses. In the future we plan to test these therapeutic interventions in combination with anti-PD-1/PD-L1 ICB therapy as a means to further activate both NK and T cell immunity to fully eradicate pancreatic cancer. This work highlights the importance of understanding the impact of the resident tumor microenvironment on anti-tumor immune responses to come up with new paradigms to potentiate immunotherapy in this and other immunologically “cold” tumor settings that are historically unresponsive to ICB therapy. Citation Format: Marcus Ruscetti. Modulating cellular senescence to reinstate natural killer cell immunity for pancreatic cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr SY24-03.

Single Cell Analyses Reveal a Non-Canonical EZH2 Activity As a Main Driver of Retinoic Acid Resistance in PLZF/Rara Leukemia

Resistance to treatment is due to the heterogeneity of the tumor which contains a subset of cancer cells that escape treatment and are responsible for the relapse. Acute Promyelocytic Leukemia (APL), the M3 subtype of AML, is a good model to illustrate these problematics. Indeed, APL driven by oncogenic fusion proteins such PML/RARA t(15;17) or PLZF/RARA t(11;17) behave differently to differentiation therapeutics. Both APLs differentiate in vivo upon Retinoic Acid (RA) treatment; however, while PML/RARA APL patients exhibit partial or complete remission, PLZF/RARA APL patients remain clinically resistant. In the present study we aim to decipher the transcriptional and epigenetic networks that is linked to t(11;17) APL resistance towards RA.We took advantage of the PLZF/RARA RA resistant murine APL model to catch relapse-initiating cell features and their vulnerabilities. By developing an integrative single-cell multi-omics analysis (scRNA-seq and scATAC-seq), we uncovered transcriptional and chromatin heterogeneity of the PLZF/RARA APL blasts. We highlighted a subset of cells insensitive to RA-induced differentiation with a strong DNA repair signature (“Rep” cluster) and exhibiting a fine tuned transcriptional network targeting the histone methyltransferase Ezh2. To validate the function of Ezh2 in APL physiology, we combined epigenomic studies with RA-treated and non-RA-treated bone marrow transplantation experiments. We revealed high Ezh2 activity that marks the relapse of RA-treated APL. However, targeting Ezh2 methyltransferase activity was not sufficient to achieve disease cure and, suggests an independent methyltransferase Ezh2 activity linked to RA resistance.These findings demonstrate the power of single-cell multi-omics integration to highlight path to sensitize therapy-resistant leukemia cells. In addition, our study uncovers a dual role of Ezh2 in APL and suggests that targeting non-canonical Ezh2 activity could be a new promising therapeutic approach for RA resistant APL. DisclosuresIwama: Nissan Chemical Corporation: Research Funding.

Synthetic Lethal Screening Identifies CHK1 Inhibition As an Exploitable Vulnerability in EZH2 Deficient T-ALL

Loss of function mutations of Enhancer of Zeste (Drosophila) homolog 2 (EZH2), the enzymatic component of Polycomb Repressive Complex 2 (PRC2), are particularly frequent in the early T cell precursor (ETP) subtype of T cell Acute Lymphoblastic Leukaemia (T-ALL), and have been associated with chemotherapy-resistance and a poor prognosis. There is thus a need to identify novel treatment strategies for this subgroup of patients.We hypothesized that inactivation of EZH2 in T-ALL cells induces specific synthetic lethal vulnerabilities that can be exploited using drugs targeting other dependency pathways, potentially resulting in a clinically meaningful therapeutic strategy. Using isogenic Jurkat T-ALL cells, with and without EZH2 -inactivating mutations generated by double-nicking CRISPR Cas9, we performed cell-based drug screening using 220 well-characterized compounds, enriched for drugs targeting epigenetic, cell cycle and DNA repair machinery. Strikingly, we observed that EZH2 -knock out (KO) cells are hypersensitive to checkpoint kinase 1 (CHK1) inhibition by the clinical-grade small molecule MK8776, showing an IC50 reduction of over 2-fold. This effect was similarly observed when testing other CHK1 inhibitors (LY2603618 and CHIR-124), but was absent in the presence of inhibitors of other DNA damage-response kinases such as ATM, ATR and CHK2, demonstrating the specificity of this synthetic lethal interaction. Furthermore, pharmacological inhibition of EZH2 methyltransferase activity by GSK126 in human T-ALL cell lines resulted in sensitization to CHK1 inhibition. Notably, MK8776 resulted in apoptosis of EZH2-deficient cells as determined by Annexin V-PI staining.CHK1 inhibition has previously been shown to result in enhanced DNA damage and accumulation of cells with incompletely replicated DNA. In line with this, EZH2 -KO cells treated with MK8776 exhibited markedly elevated levels of the DNA damage marker yH2AX. Interestingly, the highest proportion of apoptotic cells were found at the G1 phase of the cell cycle when analyzed by TUNEL-PI, suggesting that MK8776 treated EZH2 -KO cells with accumulated DNA damage override the G2 checkpoint, exit mitosis and undergo mitotic catastrophe-related cell death during G1, a feature characteristic of mitotic slippage.To investigate the mechanism further, gene expression of two EZH2 -KO Jurkat clones was compared to their isogenic parental control by RNA-seq. EZH2 -KO cells showed a striking upregulation of genes associated with human ETP-ALL including LYL1, MYCN, KIT and HHEX, a finding that could be recapitulated with GSK126 treatment. Gene set enrichment analysis (GSEA) showed significant changes of expression in MYC target genes. Importantly, MYCN protein was markedly upregulated in both EZH2 -KO and GSK126 treated cells. We speculate that increased MYCN expression induces high levels of replication stress and increased dependency on CHK1 for cell survival. Our findings suggest that specific inhibition of the CHK1 pathway can preferentially target PRC2-deficient leukemic cells, providing a potentially less toxic and more effective treatment for this high-risk subgroup of patients. DisclosuresNo relevant conflicts of interest to declare.

Targeting EZH2 as cancer therapy

Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of polycomb repressive complex 2 (PRC2) that mediate repression of target genes by trimethylation of Lys27 in histone 3 (H3K27me3). Given the reported roles of EZH2 in cancer, it is perhaps not surprising that targeting EZH2 in cancer therapy has become a hot research topic. Indeed, different types of EZH2 inhibitors are currently under development and are being evaluated by clinical trials. Recently, Murashima et al. identified NPD13668, a novel EZH2 inhibitor, by using a cell-based high-throughput screening assay. NPD13668 inhibited EZH2 methyltransferase activity, and repressed cell growth in multiple cancer cell lines, indicating a potential role for this compound in cancer treatment. In this review, we will focus on the current knowledge regarding the biological significance of PRC2 and H3K27me, and the recent advances in developing and testing drugs that target PRC2.

Abstract A47: Preclinical efficacy of CPI-1688, a novel EZH2 inhibitor, in epithelial ovarian cancer with alterations in the SWI/SNF chromatin remodeling complex

Abstract Epithelial ovarian cancer is a paradigm for exploring precision medicine in the context of genetic alteration in the SWI/SNF chromatin remodeling complex. For example, the ARID1A subunit of the SWI/SNF complex is mutated in more than 50% of clear cell ovarian carcinomas. In addition, CARM1 is amplified/overexpressed in ~20% of high-grade serous ovarian cancer. CARM1 is an arginine methyltransferase that asymmetrically dimethylates protein substrates on arginine residues. CARM1 regulates the SWI/SNF genome-wide distribution by post-transcriptionally methylating SWI/SNF core subunit BAF155. EZH2 is the catalytic subunit of the polycomb repressive complex that typically suppresses its gene expression through generating lysine 27 trimethylation on histone H3 (H3K27me3). Our previous studies established that inhibition of the methyltransferase activity of EZH2 is synthetically lethal with ARID1A mutation or CARM1 overexpression due to the antagonistic roles played by the SWI/SNF and EZH2/PRC2 complexes. Here we report the preclinical efficacy of CPI-1688, a highly selective second-generation EZH2 methyltransferase inhibitor, in ARID1A mutated clear-cell ovarian cancer cells and CARM1 overexpressed high-grade serous ovarian cancer cells. Notably, the antitumor effects conferred by CPI-1688 correlate with ARID1A mutation or CARM1 expression status. In addition, at the pharmacodynamic levels, the observed efficacy correlates with a reduction in H3K27me3 both in vitro and in vivo. This suggests that the observed antitumor effects are due to on-target inhibition of EZH2 methyltransferase activity. Finally, this observed selectivity correlates with reactivation of EZH2 target tumor suppressor genes. Together, these results indicate that CPI-1688 is a promising new therapeutic for ovarian cancer with ARID1A mutation or CARM1 overexpression. Citation Format: Elizabeth Magno, Sergey Karakashev, Jennifer Mertz, Patrick Trojer, Rugang Zhang. Preclinical efficacy of CPI-1688, a novel EZH2 inhibitor, in epithelial ovarian cancer with alterations in the SWI/SNF chromatin remodeling complex [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A47.

Evaluation of EED Inhibitors as a Class of PRC2-Targeted Small Molecules for HIV Latency Reversal.

A hallmark of human immunodeficiency type-1 (HIV) infection is the integration of the viral genome into host chromatin, resulting in a latent reservoir that persists despite antiviral therapy or immune response. Thus, key priorities toward eradication of HIV infection are to understand the mechanisms that allow HIV latency and to develop latency reversal agents (LRAs) that can facilitate the clearance of latently infected cells. The repressive H3K27me3 histone mark, catalyzed by the PRC2 complex, plays a pivotal role in transcriptional repression at the viral promoter in both cell line and primary CD4+ T cell models of latency. EZH2 inhibitors which block H3K27 methylation have been shown to act as LRAs, suggesting other PRC2 components could also be potential targets for latency reversal. EED, a core component of PRC2, ensures the propagation of H3K27me3 by allosterically activating EZH2 methyltransferase activity. Therefore, we sought to investigate if inhibition of EED would also reverse latency. Inhibitors of EED, EED226 and A-395, demonstrated latency reversal activity as single agents, and this activity was further enhanced when used in combination with other known LRAs. Loss of H3K27me3 following EED inhibition significantly increased the levels of H3K27 acetylation globally and at the HIV LTR. These results further confirm that PRC2 mediated repression plays a significant role in the maintenance of HIV latency and suggest that EED may serve as a promising new target for LRA development.

Abstract OI: Novel non-canonical functions of EZH2 in triple negative breast cancer

Abstract Patients with distant metastasis at the time of diagnosis have significantly worse prognosis. Triple negative breast carcinomas (TNBC) are heterogeneous tumors with propensity to metastasize and a guarded short term prognosis. The link between breast cancer and regulators of cell type identity was demonstrated in 2003, when enhancer of zeste homolog 2, EZH2, was reportedly overexpressed in approximately 55% of breast cancer tissue samples and was significantly associated with estrogen receptor negative status and worse disease free and overall survival. As the catalytic subunit of the Polycomb Repressive Complex 2 (PRC2), the methyltransferase EZH2 deposits trimethyl marks on histone tails of lysine 27 of histone H3 (H3K27me3) to effect transcriptional repression. However, in a subset (~ 30%) TNBC high levels of EZH2 are associated with low H3K27me3, suggesting the intriguing possibility that in this group of tumors EZH2 may promote progression through other unknown mechanisms. Recent studies have shown that in TNBC EZH2 may indeed function independent of PRC2 and H3K27me3 activity. During breast cancer initiation, EZH2 enhances breast cancer stem cells and mammary tumor development through transcriptional activation of NOTCH1 and upregulation of its signaling pathway. More recent data show that EZH2 protein binds to p38 MAP kinase with resulting EZH2 phosphorylation at threonine 367 and accumulation in the cytoplasm of TNBC cells. In breast cancer tissue samples, cytoplasmic pEZH2 T367 is significantly associated with low H3K27me3 levels and a triple negative phenotype. Mechanistically, pEZH2 T367 protein binds to cytoplasmic regulators of cell migration and invasion, and is required for metastasis. These studies demonstrate a H3K27me3-independent mechanism of EZH2 function in TNBC progression. Future investigations will be required to test the therapeutic potential of inhibiting EZH2 non-canonical mechanisms alone or in combination with pharmacological targeting of EZH2 methyltransferase activity in TNBC. Citation Format: CG Kleer. Novel non-canonical functions of EZH2 in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr OI.

EZH2 Gain-of-Function Mutations Generate a Lymphoma-Permissive Immune Niche

The polycomb histone methyltransferase protein EZH2 is highly expressed and mutated in approximately 25% of the two most common subtypes of non-Hodgkin's lymphoma: follicular lymphoma (FL) and diffuse large B cell lymphoma (DLBCL). Both of these tumor types originate from germinal center (GC) B-cells, which are highly proliferative cells that form transiently during the T-cell dependent humoral immune response. Wild type EZH2 plays a critical role in the formation of GC B-cells and mutant EZH2 drives malignant transformation of these cells in vivo. These lymphoma-associated mutations of EZH2 are gain-of-function and enhance EZH2 methyltransferase activity, which cause abnormally efficient H3K27me3, enhancing repression of transcription. Of note, EZH2 mutant lymphomas manifest aberrant suppression of antigen presentation pathways, such as loss or reduced expression of MHC-I and MHC-II, and reduced T-cell infiltrates. We show that mice engineered to conditionally express mutant Ezh2Y641F specifically in GC B-cells exhibited massive GC hyperplasia with expansion of the "light zone" -the GC niche where B-cells are selected based on the affinity of their antibodies- and extended follicular dendritic cells network. With chimeric bone marrow transplantation experiments we found that Ezh2Y641F provides a competitive advantage to activated B-cells in seeding and expanding the T-cell dependent GC reaction. Liquid chromatography separation and mass spectrometry of histone tryptic peptides revealed a massive increase in H3K27me3 in Ezh2Y641F GC B-cells, at the expense of a reduction in H3K27me2. Remarkably, we observed increased unmodified and decreased H3K27me1, indicating a loss-of-function mono-methylase activity of Ezh2Y641F. We confirmed an extensive global gain of H3K27me3 by ChIP-seq, with massive spreading of the mark both upstream and downstream TSS. RNA-seq analysis showed differential expression of genes involved in antigen processing and presentation and immune response, suggesting that B-cell selection process is altered in Ezh2Y641F GC light zone. Therefore, we hypothesized that, through an epigenetic mechanism, Ezh2 mutation makes B-cells less dependent of T-cells by aberrantly suppressing genes involved in the immune synapse. Indeed, inhibition of the interaction between TFH and GC B-cell in vivo using anti CD40L antibody, abrogated GC reaction in WT but not in Ezh2Y641F mice. Of note, EZH2 mutant GCs manifest a massive expansion of the follicular dendritic cell network in spite of lack of T-cell dependence, a finding that reflects low grade FL histology. Hence, EZH2 mutant lymphomas might progress due to acquired immune escape, which is a critical consideration when designing precision, tailored immune-therapy regimens. Disclosures Melnick: Constellation: Consultancy; Janssen: Research Funding; Epizyme: Consultancy.

PRC2 targeting is a therapeutic strategy for EZ score defined high-risk multiple myeloma patients and overcome resistance to IMiDs

BackgroundMultiple myeloma (MM) is a malignant plasma cell disease with a poor survival, characterized by the accumulation of myeloma cells (MMCs) within the bone marrow. Epigenetic modifications in MM are associated not only with cancer development and progression, but also with drug resistance.MethodsWe identified a significant upregulation of the polycomb repressive complex 2 (PRC2) core genes in MM cells in association with proliferation. We used EPZ-6438, a specific small molecule inhibitor of EZH2 methyltransferase activity, to evaluate its effects on MM cells phenotype and gene expression prolile.ResultsPRC2 targeting results in growth inhibition due to cell cycle arrest and apoptosis together with polycomb, DNA methylation, TP53, and RB1 target genes induction. Resistance to EZH2 inhibitor is mediated by DNA methylation of PRC2 target genes. We also demonstrate a synergistic effect of EPZ-6438 and lenalidomide, a conventional drug used for MM treatment, activating B cell transcription factors and tumor suppressor gene expression in concert with MYC repression. We establish a gene expression-based EZ score allowing to identify poor prognosis patients that could benefit from EZH2 inhibitor treatment.ConclusionsThese data suggest that PRC2 targeting in association with IMiDs could have a therapeutic interest in MM patients characterized by high EZ score values, reactivating B cell transcription factors, and tumor suppressor genes.

EZH2 phosphorylation by JAK3 mediates a switch to noncanonical function in natural killer/T-cell lymphoma

AbstractThe best-understood mechanism by which EZH2 exerts its oncogenic function is through polycomb repressive complex 2 (PRC2)-mediated gene repression, which requires its histone methyltransferase activity. However, small-molecule inhibitors of EZH2 that selectively target its enzymatic activity turn out to be potent only for lymphoma cells with EZH2-activating mutation. Intriguingly, recent discoveries, including ours, have placed EZH2 into the category of transcriptional coactivators and thus raised the possibility of noncanonical signaling pathways. However, it remains unclear how EZH2 switches to this catalytic independent function. In the current study, using natural killer/T-cell lymphoma (NKTL) as a disease model, we found that phosphorylation of EZH2 by JAK3 promotes the dissociation of the PRC2 complex leading to decreased global H3K27me3 levels, while it switches EZH2 to a transcriptional activator, conferring higher proliferative capacity of the affected cells. Gene expression data analysis also suggests that the noncanonical function of EZH2 as a transcriptional activator upregulates a set of genes involved in DNA replication, cell cycle, biosynthesis, stemness, and invasiveness. Consistently, JAK3 inhibitor was able to significantly reduce the growth of NKTL cells, in an EZH2 phosphorylation-dependent manner, whereas various compounds recently developed to inhibit EZH2 methyltransferase activity have no such effect. Thus, pharmacological inhibition of JAK3 activity may provide a promising treatment option for NKTL through the novel mechanism of suppressing noncanonical EZH2 activity.

Abstract 4534: Taxol therapy-induced catalytic EZH2 limits therapeutic responses in PTEN null prostate cancer

Abstract Prostate cancer (PCa) is the most common cancer and a major cause of cancer related deaths in men in North America. The phosphatase and tensin homolog gene (PTEN) is frequently inactivated in PCa. Approximately 40% of primary and 70% of metastatic PCa's have genomic alterations in the PI3K-signaling, mostly through the loss of PTEN. Majority of metastatic PCa associated with PTEN loss are treated with docetaxel; an FDA approved therapy for recurrent PCa. However, in PTEN null PCa docetaxel treatment enhances senescence and does not significantly impact tumor volume. These findings were further confirmed in PCa patients with decreased levels of PTEN. In order to understand the global impact of docetaxel in PTEN null PCa we performed an unbiased RNA-seq of prostate tissues harvested from docetaxel treated prostate specific PTEN null mice. Data analysis showed strong enrichment of chromatin modifying enzymes reactome, mainly PRC2 as one of the top ranked categories. Among them EZH2, an oncogenic methyltransferase was found to be significantly upregulated. Quantitative PCR analysis confirmed statistically significant upregulation of EZH2 in docetaxel treated mice compared to vehicle control. EZH2 upregulation was independent of androgen levels as both androgen dependent and independent cells showed striking upregulation of EZH2 when treated with docetaxel. Next, we confirmed the nature of the upregulated EZH2 (catalytic or non-catalytic) by docetaxel. EZH2 methyltransferase activity assay data showed significant induction of methyltransferase activity by docetaxel in both androgen dependent and independent conditions and in PTEN null mouse tissues. Further, we measured other methyltransferase marks i.e. H3K27me1&2, H3K4me3, H3K9me3 in the presence of docetaxel and found only specific upregulation of H3K27me3, confirming upregulation of only catalytic EZH2 by docetaxel. These data suggested a need for co-targeting EZH2 during docetaxel treatment in PTEN null PCa conditions. In order to provide a proof-of-principle we performed a tumor xenograft study in nude mice implanted with PTEN null PC3 cells. Combination of DZNeP (an EZH2 inhibitor) and docetaxel significantly inhibited tumor growth compared to each agent alone. Results obtained from the xenograft studies prompted us to perform a preclinical study in prostate specific PTEN null mice. Data obtained from this study clearly showed a significant reduction in number of high grade lesions in PTEN null tumors in combination group when compared to either docetaxel or DZNeP treated mice. We also observed clearance of senescent cells and accumulation of CD8+ T cells and classical dendritic (CD8+ Clec9a+ positive) cells. Our data suggests the necessity of designing a combination therapy of targeting EZH2 during docetaxel treatment specifically, which will likely increase the efficacy of docetaxel in PTEN null PCa. Citation Format: Mohammad Imran Khan, Abid Hamid, Vaqar Mustafa Adhami, Chloe Lang, Hasan Mukhtar. Taxol therapy-induced catalytic EZH2 limits therapeutic responses in PTEN null prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4534.

Abstract B16: Synthetic lethality by targeting EZH2 methyltransferase activity in ARID1A-mutated ovarian cancers.

Abstract ARID1A, a chromatin remodeler, shows one of the highest mutation rates across many cancer types. Notably, ARID1A is mutated in ~50% of ovarian clear cell carcinomas and ~30% of ovarian endometrioid carcinomas. To date, clinically applicable targeted cancer therapy based on ARID1A mutational status has not been described. Here we show that inhibition of the EZH2 methyltransferase acts in a synthetic lethal manner in ARID1A mutated ovarian cancer cells. ARID1A mutational status correlates with response to the EZH2 inhibitor. We identified PIK3IP1 as a direct ARID1A/EZH2 target, which is upregulated by EZH2 inhibition and contributes to the observed synthetic lethality by inhibiting PI3K/AKT signaling. Significantly, EZH2 inhibition causes regression of ARID1A mutated, but not wild-type, ovarian tumors in vivo. Together, these data demonstrate for the first time a synthetic lethality between ARID1A mutation and EZH2 inhibition. They indicate that pharmacological inhibition of EZH2 represents an urgently needed, novel treatment strategy for ARID1A mutated ovarian cancers. Citation Format: Benjamin Bitler, Ie-Ming Shih, Rugang Zhang. Synthetic lethality by targeting EZH2 methyltransferase activity in ARID1A-mutated ovarian cancers. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B16.

Abstract B30: Targeting EZH2 methyltransferase activity: A novel therapeutic approach to ARID1A-deficient cancer

Abstract Loss of function mutation of the epigenetic modulator ARID1A, a component of SWI/SNF chromatin remodeling complex, occurs in a number of cancers most notably ovarian. ARID1A is mutated in up to 60% and 30% of clear cell and endometrioid ovarian cancers, respectively. However, there currently is no effective therapy that targets ARID1A-deficient human cancers. Here we show ARID1A-deficient ovarian cancer cells are selectively sensitive to inhibition of EZH2's methyltransferase activity. EZH2 is the catalytic subunit of the polycomb repressor complex 2, which represses gene expression by generating tri-methylation epigenetic mark on lysine 27 of histone H3 (H3K27Me3). Utilizing a library of small molecules against epigenetic targets, we identified an EZH2 small molecule inhibitor that selectively inhibited the growth of ARID1A-deficient ovarian cancer cells, while exhibiting no effects in ARID1A wild-type cells. Genome-wide gene expression profile revealed that there is significant overlap of genes altered by ARID1A restoration and EZH2 methyltransferase inhibition. Specifically, the tumor suppressor and inhibitor of PI3K/AKT signaling, PIK3IP1, is regulated by both ARID1A and EZH2. Chromatin immunoprecipitation assays confirmed the occupancy of ARID1A and EZH2 at the PIK3IP1 promoter. In an intraperitoneal (IP) xenograft mouse model, EZH2 inhibitor GSK126 treatment resulted in a significant decrease in the number of IP tumor nodules. Moreover, in an independent orthotopic intra-bursal xenograft model using ARID1A wildtype and deficient cells, the EZH2 inhibitor GSK126 only suppressed the growth of ARID1A-deficient tumors. Notably, the observed effects correlated with a decrease in expression of H3K27Me3, pAKT and Ki67, a cell proliferation marker, and an increase in PIK3IP1. These data indicate loss of ARID1A and gain of EZH2 could act in epigenetic concert to promote cancer phenotypes. Taken together, these findings imply that inhibition of the methyltransferase EZH2 represents a novel therapeutic strategy for ARID1A-deficient cancers. Citation Format: Benjamin Bitler, Azat Garipov, Ie Ming Shih, Rugang Zhang. Targeting EZH2 methyltransferase activity: A novel therapeutic approach to ARID1A-deficient cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B30.

EZH2 as a therapeutic target in solid tumors

Epigenetic alterations are an important hallmark of cancer, and the enzymes that modify histone tails have emerged as attractive drug targets. The histone methyltransferase EZH2 is the catalytic subunit of PRC2, a highly conserved protein complex that regulates gene expression by methylating lysine 27 on histone H3. EZH2 is frequently overexpressed in cancer, and oncogenic gain-of-function mutations have been identified in both hematological malignancies and solid tumors. In cancer cells, the aberrant activity of the enzyme contributes to tumorigenesis by altering cell fate decisions and regulating pathways involved in proliferation, differentiation, and cell migration. Early validation efforts relied on the use of RNAi technology and non-specific small molecule inhibitors to down-regulate EZH2 and destabilize the PRC2 complex. The discovery of catalytic inhibitors of EZH2 has provided an invaluable tool for further elucidating the role of this enzyme in cancer, and preclinical studies in EZH2-mutant non-Hodgkin lymphoma have driven the clinical development of these agents. This review focuses on the use of catalytic small molecule inhibitors to identify solid tumor indications that are dependent on aberrant EZH2 methyltransferase activity. The emerging data suggests that EZH2 inhibitors will have therapeutic potential that extends beyond hematological malignancies to the solid tumor setting.