Prognostic Value of Histone Modifying Enzyme EZH2 in RCHOP-Treated Diffuse Large B-Cell Lymphoma and High Grade B-Cell Lymphoma

Background: DLBCL represent a heterogeneous group of aggressive diseases. High grade B-cell lymphomas (HGBCL) were recently individualized from DLBCL as a discrete diagnostic entity due to their worse prognosis. Currently, although most patients are successfully treated with RCHOP regimens, 1/3 will either not respond or ultimately relapse. Alterations in histone modifying enzymes have emerged as the most common alterations in DLBCL, but their role as prognostic biomarkers is controversial. We aimed to ascertain the prognostic value of EZH2 immunoexpression in RCHOP-treated DLBCL and HGBCL. Results: We performed a retrospective cohort study including 125 patients with RCHOP-treated DLBCL or HGBCL. EZH2 expression levels did not differ between diagnostic groups or between DLBCL-NOS molecular groups. We found no associations between EZH2 expression levels and outcome, including in the subgroup analysis (GC versus non-GC). Nonetheless, EZH2/BCL2 co-expression was significantly associated with worse outcome (event free survival and overall survival). Conclusion: Although EZH2 mutations are almost exclusively found in GC-DLBCL, we found similar EZH2 expression levels in both DLBCL-NOS molecular groups, suggesting non-mutational mechanisms of EZH2 deregulation. These findings suggest that the use of EZH2 antagonists might be extended to non-GC DLBCL patients with clinical benefit. EZH2/BCL2 co-expression was associated with a worse outcome.

EZH2 presents a therapeutic target for neuroendocrine tumors of the small intestine

Small intestinal neuroendocrine tumors (SI-NETs) are slow-growing tumors that seem genetically quite stable without highly recurrent mutations, but are epigenetically dysregulated. In contrast to the undetectable expression of the enhancer of zeste homolog 2 (EZH2) histone methyltransferase in the enterochromaffin cells of the small intestine, we found high and differential expression of EZH2 in primary SI-NETs and corresponding metastases. Silencing EZH2 in the SI-NET cell line CNDT2.5 reduced cell proliferation and induced apoptosis. Furthermore, EZH2 knockout inhibited tumor progression in a CNDT2.5 SI-NET xenograft mouse model, and treatment of SI-NET cell lines CNDT2.5 and GOT1 with the EZH2-specific inhibitor CPI-1205 decreased cell viability and promoted apoptosis. Moreover, CPI-1205 treatment reduced migration capacity of CNDT2.5 cells. The EZH2 inhibitor GSK126 also repressed proliferation of CNDT2.5 cells. Recently, metformin has received wide attention as a therapeutic option in diverse cancers. In CNDT2.5 and GOT1 cells, metformin suppressed EZH2 expression, and inhibited cell proliferation. Exposure of GOT1 three-dimensional cell spheroids to CPI-1205 or metformin arrested cell proliferation and decreased spheroid size. These novel findings support a possible role of EZH2 as a candidate oncogene in SI-NETs, and suggest that CPI-1205 and metformin should be further evaluated as therapeutic options for patients with SI-NETs.

LncRNA-MIAT promotes thyroid cancer progression and function as ceRNA to target EZH2 by sponging miR-150-5p

While long noncoding RNAs (lncRNAs) have been reported to play an important role in human cancer types, they remain poorly understood in papillary thyroid carcinoma (PTC). The aim of this study was to use genome-wide expression profiling to identify lncRNAs acting as competing endogenous RNAs (ceRNAs) in PTC. We constructed a ceRNA network based on our lncRNA microarray data and validated the correlation between myocardial infarction-associated transcript lncRNA (MIAT), miRNA-150-5p, and EZH2 in vitro and in vivo. We found 15 lncRNAs, 28 miRNAs, and hundreds of mRNAs involved in this ceRNA network. Splendid positive correlations were found between the MIAT and EZH2 expression in types of cancer in TCGA data. Besides, significant differences in MIAT/EZH2 expression were found among various clinicopathological features. Gain- and loss-of-function experiments revealed that MIAT inhibited cell proliferation and migration in vitro. Moreover, EZH2 was identified as a direct downstream target of miR-150-5p in PTC cells. Restoration of EZH2 expression partially abolished the biological effects of miR-150-5p. Furthermore, overexpression of MIAT was inversely correlated with miR-150-5p expression. Knockdown of MIAT produced significant behavioral alter maybe partly due to the function of the MIAT-150-5p-EZH2 network. Our findings suggest MIAT may inhibit EZH2 expression and promote PTC cell invasion via the miR-150/EZH2 pathway. Therefore, MIAT may serve as a valuable prognostic biomarker and therapeutic target for PTC.

CircTP63 promotes cell proliferation and invasion by regulating EZH2 via sponging miR-217 in gallbladder cancer

Background Gallbladder cancer (GBC) is the most common biliary tract malignancy and has a poor prognosis in patients with GBC. CircRNA TP63 (circTP63) has been implicated in cell proliferation and invasion in some tumor progress. The study aims to investigate the clinical significance and functional role of circTP63 expression in GBC. Methods The expression of circTP63 in GBC tissues or cells was detected by qRT-PCR and the association between circTP63 expression and prognosis of GBC patients was analyzed. CCK8 assay, flow cytometry analysis, transwell assay and in vivo studies were used to evaluate the cell proliferation and invasion abilities after circTP63 knockdown in GBC cells. Luciferase reporter assays and RNA pull-down assay were used to determine the correlation between circTP63 and miR-217 expression. Besides, western blot analysis was also performed. Results In the present study, we showed that circTP63 expression was upregulated in GBC tissues and cells. Higher circTP63 expression was associated with lymph node metastasis and short overall survival (OS) in patients with GBC. In vitro, knockdown of circTP63 significantly inhibited cell proliferation, cell cycle progression, migration and invasion abilities in GBC. Besides, we demonstrated that knockdown of circTP63 inhibited GBC cells Epithelial-Mesenchymal Transition (EMT) process. In vivo, knockdown of circTP63 inhibited tumor growth in GBC. Mechanistically, we demonstrated that circTP63 competitively bind to miR-217 and promoted EZH2 expression and finally facilitated tumor progression. Conclusions Our findings demonstrated that circTP63 sponged to miR-217 and regulated EZH2 expression and finally facilitated tumor progression in GBC. Thus, targeting circTP63 may be a therapeutic strategy for the treatment of GBC.

EZH2-Mediated MHC Class II Silencing Drives Immune Evasion in AML with t(16;21) (FUS-ERG)

Acute myeloid leukemia (AML) patients with the prototypical FET-ETS fusion, FUS-ERG [t(16;21)(p11;q22)], have dismal outcomes. With median event-free survival (EFS) less than 9 months, and median overall survival (OS) under 13 months (Fig. 1A), pediatric AML (pAML) with FUS-ERG has even worse outcomes than its adult counterpart (median OS: 22 months). Stem cell transplant (SCT) does not improve outcomes in FUS-ERG AML, despite predicted neoantigen formation by the fusion protein. A targetable mechanism to restore immune response and graft-versus-leukemia effect could be of considerable therapeutic benefit. Here, using blast-corrected RNA sequencing data from over 1200 patients in Children's Oncology Group (COG) trials AAML0531 and AAML1031, we find that FUS-ERG AML is distinguished from other pAML subtypes by its high expression of EZH2, a histone lysine methyltransferase for which several small molecule inhibitors have already been approved in clinical trials. The immunophenotype of FUS-ERG AML at diagnosis resembles that of post-transplant relapse with epigenetic silencing of MHC class II. FUS-ERG AML patients, regardless of age group or morphology, downregulate transcripts for MHC class II molecules (HLA-DRA, HLA-DRB1, HLA-DRB5, HLA-DPA1, HLA-DPB1, HLA-DMA, HLA-DQA1, HLA-DQB1, HLA-DMB, HLA-DOA) and the master regulator of HLA expression, CIITA (Fig. 1B). Based on MHC class II expression, we find that FUS-ERG AML at diagnosis clusters with post-SCT-relapsed adult AML, and distinct from the same adult AMLs at diagnosis (Fig. 1C; DiPersio, NEJM 2018). The mechanism of FUS-ERG driven EZH2 expression has not been previously elucidated. Signaling through the MEK/ERK/ELK1 pathway drives elevated EZH2 in some tumors, but in contrast to other pAML subtypes, EZH2 expression in FUS-ERG AML does not correlate with MEK/ERK/ELK1 pathway activation (Fig. 1D). Up to 50% of prostate cancers are driven by the TMPRSS2-ERG fusion, where EZH2 expression is induced by MYC, via downregulation of inhibitory miRNAs and binding to the EZH2 promoter. ERG itself can directly activate MYC transcription by binding of the MYC P2 promoter, creating a positive feedback loop. Based on these similarities between TMPRSS2-ERG and FUS-ERG, we hypothesized that elevated EZH2 expression in FUS-ERG AML is due to MYC activity. Indeed, we observe a strong correlation between expression of MYC and EZH2 among all pAML subsets, with FUS-ERG patients exhibiting some of the highest expression levels for each (Pearson's R=0.68) (Fig. 1E). By contrast, HNRNPH1-ERG fusions and fusions pairing FUS with other partners (FUS-X) did not yield similarly high expression of MYC or EZH2. Thus, a neomorphic combination of features from both FUS and ERG appears to drive the severe phenotype associated with this fusion. To characterize the immune-evasive phenotype presented by FUS-ERG patients at diagnosis, we conducted gene set enrichment analyses (GSEA) on sets involved in immune response. Fig. 1F shows that FUS-ERG AML is depleted of transcripts involved in "antigen processing & presentation" (p-val: 0.043) and "T cell cytotoxicity" (p-val: 0.050), as compared to other pAML. Restoration of MHC class II by inhibition of EZH2 has been shown to combat leukemic relapse. Tazemetostat, a small molecule selective and S-adenosyl methionine (SAM) competitive inhibitor of EZH2, has been approved by the FDA for the treatment of epithelioid sarcoma and follicular lymphoma; clinical trials are underway in a number of other malignancies. Given the desperate need for novel therapeutic options in these high-risk patients, we believe targeted inhibition of EZH2 may provide clinical benefit. Further preclinical investigation is currently underway in the YNH-1 FUS-ERG model. Figure 1 Figure 1. Disclosures Shaw: T-Cell and/or Gene Therapy for Cancer: Patents & Royalties.

PRMT1-mediated EZH2 methylation promotes breast cancer cell proliferation and tumorigenesis

Protein arginine methyltransferase 1 (PRMT1) is able to promote breast cancer cell proliferation. However, the detailed mechanisms of PRMT1-mediated breast cancer cell proliferation are largely unknown. In this study, we reveal that PRMT1-mediated methylation of EZH2 at the R342 site (meR342-EZH2) has a great effect on PRMT1-induced cell proliferation. We also demonstrate that meR342-EZH2 can accelerate breast cancer cell proliferation in vitro and in vivo. Further, we show that meR342-EZH2 promotes cell cycle progression by repressing P16 and P21 transcription expression. In terms of mechanism, we illustrate that meR342-EZH2 facilitates EZH2 binding with SUZ12 and PRC2 assembly by preventing AMPKα1-mediated phosphorylation of pT311-EZH2, which results in suppression of P16 and P21 transcription by enhancing EZH2 expression and H3K27me3 enrichment at P16 and P21 promoters. Finally, we validate that the expression of PRMT1 and meR342-EZH2 is negatively correlated with pT311-EZH2 expression. Our findings suggest that meR342-EZH2 may become a novel therapeutic target for the treatment of breast cancer.

EZH1/2 inhibition augments the anti-tumor effects of sorafenib in hepatocellular carcinoma

Both EZH2 and its homolog EZH1 function as histone H3 Lysine 27 (H3K27) methyltransferases and repress the transcription of target genes. Dysregulation of H3K27 trimethylation (H3K27me3) plays an important role in the development and progression of cancers such as hepatocellular carcinoma (HCC). This study investigated the relationship between the expression of EZH1/2 and the level of H3K27me3 in HCC. Additionally, the role of EZH1/2 in cell growth, tumorigenicity, and resistance to sorafenib were also analyzed. Both the lentiviral knockdown and the pharmacological inhibition of EZH1/2 (UNC1999) diminished the level of H3K27me3 and suppressed cell growth in liver cancer cells, compared with EZH1 or EZH2 single knockdown. Although a significant association was observed between EZH2 expression and H3K27me3 levels in HCC samples, overexpression of EZH1 appeared to contribute to enhanced H3K27me3 levels in some EZH2lowH3K27me3high cases. Akt suppression following sorafenib treatment resulted in an increase of the H3K27me3 levels through a decrease in EZH2 phosphorylation at serine 21. The combined use of sorafenib and UNC1999 exhibited synergistic antitumor effects in vitro and in vivo. Combination treatment canceled the sorafenib-induced enhancement in H3K27me3 levels, indicating that activation of EZH2 function is one of the mechanisms of sorafenib-resistance in HCC. In conclusion, sorafenib plus EZH1/2 inhibitors may comprise a novel therapeutic approach in HCC.

THE ROLE OF EZH2 AND ARID1A IN THE DIAGNOSIS OF FLAT UROTHELIAL LESIONS WITH ATYP

Background. Diagnosis of urothelial carcinoma in situ is of great importance because it has prognostic and therapeutic value.We aim to determine the utility of EZH2 and ARID1A as a new tool in the diagnosis of carcinoma in situ.Material and Methods. This retrospective cross-sectional study included Twenty-four specimens of flat urothelial lesions, twenty specimens of CIS, and 10 of normal adjacent urothelium that was taken by cystoscopic resection biopsy procedure. immunohistochemical expression of EZH2 and ARID1A. were evaluated in all studied cases.Results. All normal urothelium specimens showed high nuclear staining for ARID1A and negative nuclear staining for EZH2. High EZH2 expression was observed in 80 % of CIS specimens compared to 20 % of flat urothelial lesions with atypia (p=0.001 ), while high ARID1A expression was observed in 70.8 % of flat urothelial lesions with atypia compared to 25 % of CIS specimens (p=0.001). EZH2 was more accurate and specific in the diagnosis of carcinoma in situ.Conclusion. EZH2 and ARID1A are promising diagnostic markers for urothelial CIS. EZH2 is more accurate and specific than ARID1A in the diagnosis of carcinoma in situ versus other flat urothelial lesions.

Tumor-suppressive function of EZH2 is through inhibiting glutaminase

Tumors can use metabolic reprogramming to survive nutrient stress. Epigenetic regulators play a critical role in metabolic adaptation. Here we screened a sgRNA library to identify epigenetic regulators responsible for the vulnerability of colorectal cancer (CRC) cells to glucose deprivation and found that more EZH2-knockout cells survived glucose deprivation. Then, we showed that EZH2 expression was significantly downregulated in response to glucose deprivation in a glucose-sensitive CRC cell line, and EZH2-knockdown cells were more resistant to glucose deprivation. Mechanistically, EZH2 deficiency upregulated the expression of glutaminase (GLS) and promoted the production of glutamate, which in turn led to increased synthesis of intracellular glutathione (GSH) and eventually attenuated the reactive oxygen species (ROS)-mediated cell death induced by glucose deprivation. Although EZH2 functioned as an oncogene in cancer progression and EZH2 knockout abolished colorectal cancer development in a mouse model, here we revealed a mechanistic link between EZH2 and metabolic reprogramming via the direct regulation of GLS expression and observed a negative correlation between EZH2 and GLS expression in colorectal cancer tissues. These findings further confirmed the importance of heterogeneity, provided an explanation for the clinical tolerance of cancer cells to EZH2 inhibitors from the perspective of metabolism, and proposed the possibility of combining EZH2 inhibitors and glutamine metabolism inhibitors for the treatment of cancer.