MiR-138-5p Promotes HF in Rats by Reducing Histone Methylation in the Myd88 Promoter Region through Inhibition of EZH2.

BackgroundMyelodysplastic syndrome (MDS) can progress to acute myeloid leukemia (AML), and conventional chemotherapy (decitabine) does not effectively inhibit tumor cells. Enhancer of zeste homologue 2 (EZH2) and Heme oxygenase-1 (HO-1) are two key factors in patients resistance and deterioration.MethodsIn total, 58 MDS patients were divided into four groups. We analyzed the difference in HO-1 and EZH2 expression among the groups by real-time PCR. After treatment with Hemin or Znpp IX, flow cytometry was used to detect apoptosis and assess the cell cycle distribution of tumor cells. Following injection of mice with very high-risk MDS cells, spleen and bone marrow samples were studied by immunohistochemistry (IHC) and hematoxylin and eosin (H&E) staining. MDS cells overexpressing EZH2 and HO-1 were analyzed by high-throughput sequencing. The effect of HO-1 on the pRB-E2F pathway was analyzed by Western blotting. The effects of decitabine on P15INK4B and TP53 in MDS cells after inhibiting HO-1 were detected by Western blotting.ResultsReal-time PCR results showed that EZH2 and HO-1 expression levels were higher in MDS patients than in normal donors. The levels of HO-1 and EZH2 were simultaneously increased in the high-risk and very high-risk groups. Linear correlation analysis and laser scanning confocal microscopy results indicated that EZH2 was related to HO-1. MDS cells that highly expressed EZH2 and HO-1 infiltrated the tissues of experimental mice. IHC results indicated that these phenomena were related to the pRB-E2F pathway. High-throughput sequencing indicated that the progression of MDS to AML was related to EZH2. Using the E2F inhibitor HLM006474 and the EZH2 inhibitor JQEZ5, we showed that HO-1 could regulate EZH2 expression. HO-1 could stimulate the transcription and activation of EZH2 through the pRB-E2F pathway in MDS patients during chemotherapy, which reduced TP53 and P15INK4B expression.ConclusionsEZH2 was associated with HO-1 in high-risk and very high-risk MDS patients. HO-1 could influence MDS resistance and progression to AML.

Biomarkers that predict prostate cancer (PCa) adaptation to hormone therapy are essential for developing novel therapeutic strategies to manage castration-resistant PCa (CRPC) progression. Enhancer of zeste homolog 2 (EZH2) methyltransferase, the enzymatic component of polycomb repressor complex 2 (PRC2), mediates histone 3 lysine 27 trimethylation (H3K27me3) to represses target genes. Elevated EZH2 levels and decreased PRC2 target gene expression predict poor prognosis in PCa. To test whether altered EZH2 expression and H3K27me3 mark intensity are linked to CRPC progression, we analyzed these parameters in matched primary PCa and lymph node metastasis (LN-mets) from 18 therapy naïve (TN) and neo-adjuvant hormone therapy (NHT) -treated patients. EZH2 expression was observed at varying intensities in all adenocarcinoma samples. Median EZH2 expression did not differ between TN and NHT-treated prostate tumors, but was enriched in discrete foci of varying densities in TN prostatectomy specimens while being uniformly expressed in NHT-treated specimens. In LN-mets from TN patients, EZH2 expression was uniform and reflected the predominant expression pattern observed in matched prostatectomy specimens. In the NHT-treated LN-met cohort, EZH2 expression was localized to discrete foci, however its median expression was reduced relative to the TN LN-met cohort and to both primary PCa cohorts. In contrast, H3K27me3 intensity was uniform in all specimens and on average was indistinguishable between primary PCa and LN-mets irrespective of treatment. Median EZH2 expression in patient-matched prostatectomy and LN-met specimens was directly correlated in both TN and NHT-treated cohorts. H3K27me3 intensity levels were not correlated in patient-matched prostatectomy and LN-met specimens from TN patients, but were correlated in the NHT-treated cohort. While no correlation was observed for EZH2 expression and H3K27me3 intensity within each specimen, the ratio of H3K27me3 intensity to EZH2 expression was increased in the NHT-treated LN-met cohort relative to the TN LN-met cohort and to either primary PCa cohort. This effect was driven by the decreased EZH2 expression in the LN-mets from NHT-treated patients. We conclude that EZH2 expression in LN-mets is predicted by its expression in foci of the patient-matched primary tumors from both TN and NHT-treated patients. This correlation is not observed for H3K27me3 intensity levels of TN primary tumors and LN-mets, but is strongly correlated in NHT-treated specimens. NHT treatment appears to suppress EZH2 expression in LN-mets without impacting H3K27me3 intensity levels. These results suggest that while EZH2 expression and PRC2 activity may drive phenotypic changes that promote oncogenic potential, persistence of this epigenetic signature is a better predictor of CRPC progression. Citation Format: Mitali Pandey, Ladan Fazli, Antonio Hurtado-Coll, Martin E. Gleave, Michael E. Cox. Predictive significance of EZH2 expression and H3K27me3 intensity in therapy-naïve and neoadjuvant therapy-treated metastatic prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5359. doi:10.1158/1538-7445.AM2013-5359

Endometrial cancer (EC) incidence is increasing globally, highlighting the need for novel therapies targeting molecular drivers of malignancy. Enhancer of zeste homolog 2 (EZH2), a histone methyltransferase implicated in tumor progression, is overexpressed in EC; however, its precise role and therapeutic potential remain unclear. In this study, we aimed to investigate EZH2 expression, its functional role, and the efficacy of EZH2 inhibitors in EC cell lines. EZH2 expression was analyzed in eight EC cell lines using western blotting and immunocytochemistry. The efficacy of five EZH2 inhibitors (CPI-1205, EI1, EPZ005687, EPZ-6438, and GSK126) was evaluated using drug sensitivity assays. Furthermore, functional analyses, including cell proliferation, colony formation, migration, and invasion assays, were performed following siRNA-mediated EZH2 knockdown in HEC-50B cells. Variable EZH2 expression was observed across EC cell lines, with high levels in HEC-50B and Ishikawa 3-H-12 cells. EZH2-high expressing cell lines were markedly more sensitive to EZH2 inhibitors, particularly GSK126, compared to EZH2-low expressing lines. EZH2 knockdown in HEC-50B cells reduced EZH2 expression and decreased sensitivity to EZH2 inhibitors, confirming target specificity, while also attenuating cell proliferation, colony formation, migration, and invasion. EZH2 plays a crucial role in promoting malignant phenotypes in EC, and its expression level correlates with cellular sensitivity to EZH2 inhibitors. These findings suggest that EZH2 could serve as a valuable therapeutic target and predictive biomarker for personalized medicine in EC.

To investigate the effect and mechanism of histone methyltransferase enhancer of zeste homolog 2 (EZH2) on sepsis-induced T cell dysfunction. Twenty-four male C57BL/6 mice were divided into three groups randomly: sham operated group, sepsis model group [cecum ligation and puncture (CLP)+dimethyl sulfoxide (DMSO) group] and EZH2 selective inhibitor treated group (CLP+GSK126 group), with 8 mice in each group. Sepsis murine model was reproduced by CLP. CLP+DMSO group and CLP+GSK126 group were treated with DMSO or GSK126 (10 mg/kg) respectively right after surgery through intraperitoneal injection. The mice were sacrificed 24 hours after operation, and the mesenteric lymph nodes were collected. The expression of EZH2, apoptosis rates, cell proliferation marker ki-67 antigen positive T lymphocytes (ki-67+ cell), interferon-γ positive T lymphocytes (IFN-γ + cell), programmed death receptor-1 positive T lymphocytes (PD-1+ cell) and programmed death-ligand 1 positive T lymphocytes (PD-L1+ cell) were determined by flow cytometry. Compared with sham operated group, the expression of EZH2 in T lymphocytes was up-regulated on mesenteric lymph nodes of CLP+DMSO group. Compared with CLP+DMSO group, the ratio of CD3+ T lymphocytes in CLP+GSK126 group was up-regulated (0.70±0.02 vs. 0.50±0.07, P < 0.01), indicating that the EZH2 inhibitor could increase the number of T lymphocytes in lymph nodes of septic mice; the ratio of ki-67+ cells in CD4+ and CD8+ T lymphocytes in CLP+GSK126 group was increased (CD4+: 0.74±0.05 vs. 0.63±0.04, CD8+: 0.82±0.06 vs. 0.70±0.04, both P < 0.05), indicating that the EZH2 inhibitor could increase the ratio of T lymphocytes with high proliferative activity in lymph nodes of septic mice. However, no significant difference was found on both CD4+ and CD8+ T lymphocytes apoptosis rates in the mesenteric lymph nodes of mice between CLP+GSK126 group and CLP+DMSO group [CD4+: (21.53±2.87)% vs. (20.48±3.21)%, CD8+: (8.34±1.02)% vs. (7.71±1.38)%, both P > 0.05], indicating that no extra T lymphocytes apoptosis was induced by EZH2 inhibitor. Compared with CLP+DMSO group, the ratios of IFN-γ + CD4+ and IFN-γ + CD8+ T lymphocytes were increased in CLP+GSK126 group (IFN-γ +CD4+: 0.31±0.11 vs. 0.14±0.06, IFN-γ +CD8+: 0.30±0.10 vs. 0.13±0.06, both P < 0.05), suggesting that secretion of IFN-γ in lymph nodes by sepsis T lymphocytes was augmented after EZH2 inhibitor administration. Furthermore, compared with CLP+DMSO group, the ratio of PD-1+ cell in CD8+ T lymphocyte was down-regulated in CLP+GSK126 group (0.092±0.006 vs. 0.135±0.004, P < 0.01), suggesting that EZH2 inhibitor restrained the PD-1 expression on sepsis lymphoid node CD8+ T lymphocytes, however, it had no significant effect on PD-L1+ cells. EZH2, regulates sepsis-induced T lymphocyte dysfunction, possibly through modulating the expression of PD-1.

A Positive Correlation Between the EZH2 and PD-L1 Expression in Resected Lung Adenocarcinomas

Background: The molecular mechanisms governing anti-PD-1 resistance in melanoma are not fully understood. Suboptimal transcriptional and molecular response to interferon-gamma (IFNγ) is commonly identified in tumors with intrinsic or acquired resistance to anti-PD-1. Epigenetic regulators have been shown to reprogram IFNγ responses in some cases, though the mechanism is not clear. The ability of melanoma cells to express MHC-II is a positive clinical predictor to anti-PD-1 therapy, and MHC-II is induced only on cells with robust IFNγ responses. Enhancer of zeste homolog 2 (EZH2), an epigenetic regulator with both methyltransferase-dependent and -independent function, can suppress IFNγ target genes and is frequently overexpressed in melanoma. However, the mechanisms whereby EZH2 interacts with the IFNγ pathway and its role in melanoma-specific MHC-II expression is not known. Methods: HLA-DR (MHC-II)-proficient (A375, SKMEL28 and SKMEL5) and -deficient (CHL-1 and MEWO) melanoma cell lines were stimulated with IFNγ for up to 48h, and the modulation of EZH2 protein and function were studied temporally. To test the role of EZH2 in MHC-II (HLA-DR) expression in IFNγ responses, genetic (siRNA) and chemical (GSK343) EZH2 inhibition was utilized. Results: EZH2 was highly expressed in all cell lines tested, regardless of HLA-DR-proficiency. In 2 out of 3 HLA-DR-inducible melanoma cell lines, EZH2 protein expression was downregulated (4-24 hrs) in response to IFNγ stimulation, without effects on EZH2 mRNA expression or downstream tri-methylated H3K27 expression. Blocking proteosomal degradation with MG-132 reversed the IFNγ-induced decrease in EZH2 expression in HLA-DR-proficient cell lines. After siRNA knockdown of EZH2, SKMEL28 cells increased constitutive HLA-DR expression, while A375 cells became more sensitive to IFNγ-induced HLA-DR upregulation. In addition, IFNγ-induced HLA-DR was increased in A375 cells with EZH2 inhibition using GSK343. Conclusion: This work demonstrates a potential role for EZH2 in suppressing IFNγ responses, including the induction of MHC-II, in melanoma cells. EZH2 is subject to proteosomal degradation in MHC-II-inducible cell lines, which, in part, contributes to the expression of MHC-II. Citation Format: Jamaal L. James, Susan R. Opalenik, Abigail Toren, Rebecca S. Cook, Justin M. Balko. IFNγ signaling drives EZH2 degradation to induce MHC-II expression in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4728.

Background: The molecular mechanisms governing anti-PD-1 resistance in melanoma are not fully understood. Suboptimal transcriptional and molecular response to interferon-gamma (IFNγ) is commonly identified in tumors with intrinsic or acquired resistance to anti-PD-1. Epigenetic regulators have been shown to reprogram IFNγ responses in some cases, though the mechanism is not clear. The ability of melanoma cells to express MHC-II is a positive clinical predictor to anti-PD-1 therapy, and MHC-II is induced only on cells with robust IFNγ responses. Enhancer of zeste homolog 2 (EZH2), an epigenetic regulator with both methyltransferase-dependent and -independent function, can suppress IFNγ target genes and is frequently overexpressed in melanoma. However, the mechanisms whereby EZH2 interacts with the IFNγ pathway and its role in melanoma-specific MHC-II expression is not known. Methods: HLA-DR (MHC-II)-proficient (A375, SKMEL28 and SKMEL5) and -deficient (CHL-1 and MEWO) melanoma cell lines were stimulated with IFNγ for up to 48h, and the modulation of EZH2 protein and function were studied temporally. To test the role of EZH2 in MHC-II (HLA-DR) expression in IFNγ responses, genetic (siRNA) and chemical (GSK343) EZH2 inhibition was utilized. Results: EZH2 was highly expressed in all cell lines tested, regardless of HLA-DR-proficiency. In 2 out of 3 HLA-DR-inducible melanoma cell lines, EZH2 protein expression was downregulated (4-24 hrs) in response to IFNγ stimulation, without effects on EZH2 mRNA expression or downstream tri-methylated H3K27 expression. Blocking proteosomal degradation with MG-132 reversed the IFNγ-induced decrease in EZH2 expression in HLA-DR-proficient cell lines. After siRNA knockdown of EZH2, SKMEL28 cells increased constitutive HLA-DR expression, while A375 cells became more sensitive to IFNγ-induced HLA-DR upregulation. In addition, IFNγ-induced HLA-DR was increased in A375 cells with EZH2 inhibition using GSK343. Conclusion: This work demonstrates a potential role for EZH2 in suppressing IFNγ responses, including the induction of MHC-II, in melanoma cells. EZH2 is subject to proteosomal degradation in MHC-II-inducible cell lines, which, in part, contributes to the expression of MHC-II. Citation Format: Jamaal L. James, Susan R. Opalenik, Abigail Toren, Rebecca S. Cook, Justin M. Balko. IFNγ signaling drives EZH2 degradation to induce MHC-II expression in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4728.

Background. The molecular events associated with NSCLC pathogenesis and tumor progression need to be better elucidated. The enhancer of zeste homolog 2 (EZH2) is a DNA methyl transferase involved in malignant transformation and tumor progression of several human carcinomas, including lung. We investigated EZH2 expression by immunohistochemistry (IHC) in the early pathogenesis of NSCLC and progression in a large series of clinically well-annotated tissue specimens. Methods. We examined by IHC nuclear EZH2 expression using formalin-fixed and paraffin-embedded tissue specimens obtained from surgically resected tumors in tissue microarrays (TMAs) including: a) stage I-III NSCLC tumors (SCCs, n=272; adenocarcinomas, n=456); b) paired primary tumors and brain metastases (n=70); and, c) bronchial preneoplastic squamous lesions (n=51) and mildly abnormal/normal bronchial epithelia (n=203). In stage I-III tumors, we correlated EZH2 expression with clinico-pathological features, including patients’ recurrence-free survival (RFS), and overall survival (OS), in a subset of these tumors, with IHC expression of 80 proteins and EGFR and KRAS mutation status. Results. EZH2 expression was significantly (P&amp;lt;0.0001) higher in SCC (mean score=128.6) compared to adenocarcinoma (mean score=56.8). In adenocarcinoma, higher EZH2 expression significantly correlated with ever-smoking status (P&amp;lt;0.0001) and less differentiated histology features (solid histology pattern; P&amp;lt;0.0001). In multivariate analysis, for adenocarcinoma patients, higher EZH2 expression, as a continuous variable, associated with significantly worse RFS (HR 1.006 95%CI 1.0-1.011; P=0.03) and OS (HR 1.004 95%CI 1.0-1.009; P=0.03). In publicly available array datasets of lung adenocarcinoma patients, high EZH2 mRNA correlated with worse RFS and OS. NSCLC brain metastases showed significantly (P=0.0004) higher EZH2 expression than corresponding primary tumors. In bronchial epithelia, normal and hyperplastic cells demonstrated low levels of EZH2 expression; significantly higher expression was associated with increasing severity of squamous dysplastic changes (P&amp;lt;0.0001). In NSCLC tumors, EZH2 expression positively correlated (P&amp;lt;0.0001) with IHC expression of Ki67, FEN1, and UBE2C. In lung adenocarcinomas, EGFR-mutant tumors showed significantly lower EZH2 expression than wild-type tumors. Conclusions. Our findings indicate that EZH2 is frequently expressed in NSCLC, particularly in poorly differentiated adenocarcinomas. In adenocarcinomas, EZH2 associates with worse patient outcomes. These data suggest that EZH2 expression represents an early event in NSCLC pathogenesis and associates with tumor progression and metastasis, representing a novel target for chemoprevention and therapeutic strategies. Supported by DoD grants W81XWH-04-1-0142 and DoD W81XWH-07-1-0306. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3196. doi:10.1158/1538-7445.AM2011-3196

Purpose: Colorectal cancer (CRC) is the second most commonly diagnosed cancer and the third leading cause of cancer mortality in Japan. Almost 15% and 10% of patients with CRC are diagnosed with synchronous and metachronous liver metastasis, respectively. In the last decade, the therapeutic strategies including chemotherapy and molecular targeted therapy have improved. However, the prognosis of patients with CRC remains poor because of their acquirement of chemoresistance. Previous studies have shown that enhancer of zeste homologue 2 (EZH2) is associated with proliferation of various cancers. EZH2 is the catalytic subunit of Polycomb repressive complex2 (PRC2) which catalyzes the methylation of lysine 27 on histone H3 (H3K27), leading to repress the transcription of tumor-suppressor genes. In addition, high EZH2 expression is associated with the resistance to cisplatin in non-small cell lung carcinoma. However, the association between the resistance to oxaliplatin and the expression of EZH2 is unrevealed. The aim of this study is to investigate the correlation between the expression of EZH2 and response to oxaliplatin-based chemotherapy as well as survival of patients with colorectal liver metastasis (CRLM). Experimental Design: Sixty-one patients who underwent first-line oxaliplatin-based chemotherapy before resection of CRLM were enrolled in this study. Immunohistochemistry of EZH2 was performed, and then the correlation between response to chemotherapy and survival was evaluated. Results: Immunohistochemistry revealed 38 patients (62.3%) with high EZH2 expression, and 23 patients (37.7%) with low EZH2 expression. Of the 38 patients with EZH2 high expression, 15 patients (39.5%) responded to oxaliplatin-based chemotherapy with either complete response or partial response. Of the 23 patients with low EZH2 expression, 14 patients (60.9%) exhibited a response to chemotherapy. There was significant difference in response to oxaliplatin-based chemotherapy between high and low EZH2 expression patients (p = 0.04). Univariate survival analysis indicated that patients with high EZH2 expression had a lower overall survival than those with low EZH2 expression. Moreover, multivariate Cox regression analysis revealed that high EZH2 expression was an independent prognostic factor for overall survival. Kaplan-Meier survival curves also confirmed that high EZH2 expression correlates with poor prognosis in patients who underwent hepatectomy for CRLM. Conclusions: Our results indicated that prognosis of patients with high EZH2 expression who received preoperative oxaliplatin-based chemotherapy and underwent hepatectomy for CRLM was worse than those with low EZH2 expression. EZH2 may be a predictive factor for oxaliplatin-based chemotherapy response and overall survival in patients with CRLM. Citation Format: Mayuko Ohuchi, Yasuo Sakamoto, Ryuma Tokunaga, Kenichi Nakamura, Yuki Kiyozumi, Daisuke Izumi, Keisuke Kosumi, Kazuto Harada, Junji Kurashige, Yukiharu Hiyoshi, Shiro Iwagami, Yoshifumi Baba, Yuji Miyamoto, Naoya Yoshida, Hideo Baba. Prognostic impact of enhancer of zeste homologue 2 (EZH2) in patients underwent hepatectomy for colorectal liver metastases who received preoperative oxaliplatin-based chemotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2277. doi:10.1158/1538-7445.AM2015-2277

Simple SummaryAdult T-cell leukemia/lymphoma (ATLL) is a highly aggressive mature T-cell neoplasm with an extremely poor prognosis. For decades, the first-line therapy for ATLL has been CHO(E)P, although treatment refractoriness and relapse are common. Therefore, there is a need to search for new treatment options. This study, focusing on EZH2, an important epigenetic regulator, and associated intracellular signaling molecules, demonstrates that both EZH2 and pERK expression are upregulated in ATLL, contributing to tumor aggressiveness in biopsied patients’ tumor tissues. Their inhibitors could be potential therapeutic targets for these aggressive T-cell neoplasms.EZH2, a subunit of the polycomb repressive complex 2 (PRC2), is an important methyltransferase that catalyzes the trimethylation of histone H3 at lysine 27 (H3K27me3). EZH2 is overexpressed in various malignancies. Here, we investigated EZH2 expression and potential signaling molecules that correlate with EZH2 expression in ATLL and other T-cell neoplasms. Immunohistochemical staining (IHC) was performed for EZH2, pERK, MYC, and pSTAT3 on 43 ATLL cases and 104 cases of other T-cell neoplasms. Further IHC studies were conducted for Ki-67, SUZ12, and H3K27me3 on ATLL cases. All ATLL cases showed EZH2 overexpression. In other T-cell neoplasms, a high prevalence of EZH2 overexpression was identified (86%), except for T-PLL (33%). In ATLL, EZH2 overexpression correlated with pERK co-expression (86%), while only a small subset of cases showed MYC (7%) or pSTAT3 (14%) co-expression. In the other T-cell neoplasms, there was a variable, but higher, co-expression of EZH2 with pERK, MYC, and pSTAT3. In ATLL, enhanced EZH2 expression correlated with higher Ki-67 staining, SUZ12 (another PRC2 subunit), and H3K27me3 co-expression. In conclusion, EZH2 is overexpressed in ATLL and is associated with pERK expression. It correlates with an increased proliferation index, indicating an aggressive clinical course. EZH2 also correlates with SUZ12 and H3K27me3 co-expression, suggesting its PRC2-dependent catalytic activity through trimethylation. Additionally, EZH2 is overexpressed in most T-cell neoplasms, suggesting that EZH2 could function as an oncogenic protein in T-cell tumorigenesis. EZH2 and pERK could serve as potential therapeutic targets for treating aggressive ATLL. EZH2 could also be targeted in other T-cell neoplasms.

[Background and Aims]Enhancer of Zeste Homolog 2 (EZH2) is one of the polycomb proteins and involved in the progress of several solid tumors by methylating histone3 Lysin27 (H3K27) and silencing various genes. However, the role of EZH2 in cholangiocarcinoma has not been clarified. In this study, we investigated the significance of EZH2 expression in intrahepatic and extrahepatic cholangiocarcinoma progression.[Patients and Methods]Fourty-seven intrahepatic cholangiocarcinoma (ICC) patients and 45 extrahepatic cholangiocarcinoma (ECC) patients treated with curative resection from 1993 to 2010 were enrolled. We examined the EZH2 expression by immunohistochemistry, and relationship between EZH2 expression and clinicopathological factors, such as gender, age, CA19-9, tumor diameter, tumor number, histological vascular invasion, differentiation, lymph node metastasis and prognosis. To assess whether EZH2 relates to angiogenesis and tumor growth, Ki67 expression and microvessel density (MVD) using CD34 were also examined by immunohistochemistry. EZH2 expression was investigated eight cholangiocarcinoma cell lines. Using small interfering RNA against EZH2, the influence of EZH2 knock-down on cholangiocarcinoma cell proliferation and invasion was studied in vitro. [Results]In ICC, 24 patients (51.0%) shows a high expression of EZH2 when the cutoff value was determined to be 10%. The EZH2 high expression is significantly correlated with tumor diameter &amp;gt; 3.5cm (P=0.0113) and MVD score (P=0.039). The high EZH2 expression group showed significantly worse overall survival (p=0.045). In ECC, 19 patients(42.2%) showed a high EZH2 expression. The EZH2 high expression is significantly correlated with positive lymph node metastasis (p=0.0469), high Ki67 expression (P=0.0017). The high EZH2 expression group showed significantly worse overall survival (P=0.0261). All of the eight cholangiocarcinoma cell lines expressed EZH2 in a various level. When we knock down the expression of EZH2 in RBE cells, the expression of H3K27 was reduced and cell growth decreased. However, EZH2 down-regulation didn't affect cell invasion.[Conclusion]EZH2 may relate to the progression of intra and extrahepatic cholangiocarcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1055. doi:1538-7445.AM2012-1055

350 Background: In prostate cancer, overexpression of EZH2 is associated with disease recurrence, androgen independent growth, cell invasion, and metastases. We evaluated the prognostic significance of EZH2 gene expression in metastatic biopsies from men with CRPC. Methods: EZH2 (RNA) expression was evaluated in two serial metastatic site biopsies in patients (pts) prior to treatment with abiraterone acetate and prednisone (pre-AA/P) and after 12 weeks of treatment (post-AA/P). All pts were enrolled and prospectively followed for clinical outcomes. The primary endpoint was overall survival (OS), defined as the time from CRPC to death or last follow-up. Cox proportional hazard regression analysis was performed on EZH2 expression using normalized fragments per kilobase million (FPKM) &gt; 1 for association with OS and time to treatment failure (TTF). Results: Of 92 enrolled pts, 59 pts had (pre-AA/P) gene expression using RNA-seq, 45 had paired pre and post-AA/P RNA-seq available for analysis. In pre-AA/P samples, the median EZH2 expression was 4.82 FPKMs (range 1.05-38.8). Elevated expression of EZH2 was associated with shorter OS [hazard ratio (HR) 2.58, p = 0.016]. Radiographic progression at 12 weeks was more frequent in high EZH2 tumors compared to low EZH2 tumors (48% vs 20%, p = 0.03, Fisher’s exact test) No statistically significant associations were identified between EZH2 expression and TTF. In paired post-AA/P biopsies, EZH2 expression increased in 13/45 tumors and decreased in 32/45 tumors. The median log2 fold change in EZH2 expression was -1.38 (range -6.7 to 3.1). In post-AA/P biopsies, no associations were identified between an increase in EZH2 expression and OS or TTF. Conclusions: EZH2 expression represents a potential novel prognostic biomarker in mCRPC and warrants further exploration. Clinical trial information: 01953640.

Multiple reciprocal interactions within B-cell receptor and AKT signaling pathways regulate response to EZH2 inhibition in germinal center-derived diffuse large B-cell lymphoma

Histone methyl transferases (HMTs) and demethylases are chromatin modifying enzymes known to play a key role in establishing and maintaining chromatin structure and thereby contributing to the control of gene expression. The histone methyltransferase Enhancer of Zeste Homologue 2 (EZH2) is the catalytic component of the Polycomb Repressive Complex 2 and mediates trimethylation of lysine 27 on histone 3 (H3K27me3), which correlates with transcriptional repression. EZH2 has been widely implicated in cancer and inhibition of its catalytic activity recently emerged as a novel therapeutic approach to treat human cancers. Constellation has developed potent, selective and reversible EZH2 small molecule inhibitors that are currently being tested in clinical trials. We have previously reported EZH2 dependencies across non-Hodgkin Lymphoma subtypes, including models harboring both wild-type and mutant EZH2. To identify other cancer types that may rely on EZH2 for survival, we carried out long term growth assays across a 200+ cancer cell line panel. We observed that over 50% of multiple myeloma cell lines show -time and -dose dependent phenotypic response to EZH2 inhibition. Similar to lymphoma, EZH2 inhibitors induce apoptosis after continuous treatment over a longer time period. To understand the underlying molecular consequences of EZH2 inhibition in multiple myeloma, we performed RNA-sequencing and ChIP-sequencing in the absence and presence of the inhibitor. We identified an EZH2-controlled transcriptional signature across various multiple myeloma models and key downstream effectors including CDKN1A in individual models. EZH2 inhibitors such as CPI-169 achieve tumor growth inhibition in several multiple myeloma subcutaneous xenograft models at well tolerated doses, and this impact on tumor growth correlated well with target inhibition. To expand the scope of EZH2 inhibitor application in multiple myeloma, we systematically combined EZH2 inhibitors with standard of care agents, including, lenalidomide, prednisolone, bortezomib and HDAC inhibitors. We observed synergy of EZH2 inhibitors with several of these agents in vitro and in vivo and are currently exploring the molecular basis of these combinatorial effects. In conclusion, we provide ample evidence suggesting multiple myeloma as a disease indication in which EZH2 inhibitors may show clinical benefit as a single agent and in combination with approved therapeutics. Citation Format: Shilpi Arora, Kaylyn Williamson, Srividya Balasubramanian, Jennifer Busby, Shivani Garapaty-Rao, Charlie Hatton, Dhanalakshmi Sivanandhan, Barbara Bryant, Emmanuel Normant, Patrick Trojer. EZH2 inhibitors reveal broad EZH2 dependencies in multiple myeloma. [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 PR09.

The authors previously observed that enhancer of zeste homolog 2 (EZH2) overexpression was associated significantly with the development of oral cancer. In the current study, they investigated whether EZH2 can function as a prognostic predictor for patients with head and neck squamous cell carcinoma (HNSCC). Expression levels of EZH2 in HNSCC cells were detected using reverse transcriptase-polymerase chain reaction (PCR) and Western blot analyses. In addition, the effects of EZH2 ablation on the proliferation and invasion of HNSCC cells were investigated through small interfering RNA (siRNA)-mediated knockdown. Real-time PCR and immunohistochemistry were used to evaluate EZH2 and cyclin D1 expression in 46 HNSCC samples, and the expression levels also were re-evaluated in 124 independent samples by immunohistochemistry. EZH2 expression was elevated remarkably in HNSCC specimens and cell lines. Upon EZH2 silencing, the proliferation and invasion of HNSCC cells were remarkably suppressed. EZH2 expression frequently was correlated with cyclin D1 expression (P = .034) and tumor differentiation (P = .020). In addition, both EZH2 messenger RNA levels and EZH2 protein levels were strongly associated with signs of histologic severity (P = .012 and P = .032, respectively). Univariate analysis revealed that high EZH2 expression was associated with worse overall survival (P = .001) and disease-free survival (P = .002). The combined expression of EZH2 and cyclin D1 had superior prognostic ability for patients with HNSCC than the expression of either marker alone. In multivariate analysis, EZH2 expression was identified as an independent predictor of overall and disease-free survival. The current results indicated that EZH2 is an independent prognostic indicator for patients with HNSCC. In addition, an analysis of the combined expression of EZH2 and cyclin D1 can serve as a more powerful prognostic predictor for patients with HNSCC.