Enhancer Of Zeste Homolog 2 Research Articles

EZH2 inhibition dampens autoantibody production in lupus by restoring B cell immune tolerance.

The aim of this study was to elucidate the role of enhancer of zeste homolog 2 (EZH2) in the breakdown of B cell immune tolerance and production of autoantibodies in systemic lupus erythematosus (SLE), and to explore the therapeutic effects of EZH2 inhibition on lupus. Peripheral blood mononuclear cells (PBMCs) were collected from new-onset SLE patients for flow cytometric analysis. Pristane-induced lupus mice were constructed, and the EZH2 inhibitor was administrated by intraperitoneal injection to treat lupus mice. Blood and urine were collected from lupus mice to detect autoantibodies and proteinuria, and renal pathology scores were assessed. Mouse spleen B cells were sorted with magnetic beads and subjected to flow cytometric apoptosis detection, real time quantitative PCR (RT-qPCR), and western blotting (WB). EZH2 expression was elevated in diverse B-cell subsets in both SLE patients and pristane-induced lupus mice. The EZH2 inhibitor attenuated lupus-like symptoms and dampened autoantibody production in pristane-induced lupus mice. Inhibition of EZH2 also reduced autoantibody secretion by plasma cells from lupus patients. Mechanistically, EZH2 mediated the impaired apoptosis of autoreactive B cells and the differentiation of autoantibody producing plasma cells by inhibiting multiple cyclin-dependent kinase inhibitor (CKI) genes. EZH2 mediated the breakdown of B-cell peripheral immune tolerance by inhibiting CKI genes and participated in the generation of autoantibodies in SLE. EZH2 inhibition could serve as a promising drug intervention for the treatment of SLE.

Combined inhibition of EZH2 and histone deacetylases as a potential epigenetic therapy for human uterine sarcoma cells.

e17620 Background: Uterine sarcoma is strongly associated with poor prognosis, high rates of recurrence, and metastasis. However, the molecular mechanisms underlying the origin and development of uterine sarcoma and treatment options are limited. Enhancer of zeste homolog 2 (EZH2) and histone deacetylase (HDACs) play an important role in cancer progression. This study aimed to assess the effect of targeting both EZH2 and HDACs on the phenotype of uterine sarcoma (US) cells. Methods: Tazemetostat (EPZ-6438, E7438), a potent, and selective EZH2 inhibitor, was first approved by the US Food and Drug Administration (FDA) in 2020. Entinostat (MS-275, SNDX-275) is one of the strong inhibitors for HDAC1 and HDAC3, Vorinostat is used to treat cutaneous T-cell lymphoma, Tucidinostat (Chidamide, HBI-8000, CS-055) is a low nanomolar inhibitor of HDAC1, 2, 3, and 10. Uterine sarcoma cell line (MES-SA) was treated with different concentrations (1.56-200 mM) of Tazemetostat and/or Entinostat, Vorinostat, Tucidinostat (Chidamide). for 24, 48, 72 h, respectively. Further mechanistic confirmation was carried out on best combination in both 2D and 3D culturing using IHC and real time PCR. Results: Our data showed that Entinostat synergistically (with a combination index 0.539) augments the cytotoxic activity of Tazemetostat in both 2D and spheroids in vitro model systems. The IC50 values for Tazemetostat were significantly reduced to 4.5 mM after combination treatment with 6.25 mM Entinostat for 48 h. Entinostat enhanced the antiproliferative effect of Tazemetostat with a decrease in the expression of proliferative markers Ki67(P < 0.0001) and PCNA (P < 0.01). Furthermore, combination treatment significantly increased US cell apoptosis, concomitantly with increased both BAX and Caspase-3 levels and decreased Bcl-2 (P < 0.0001) using 2D and spheroid in vitro model (P < 0.001). The finding was confirmed by IHC analysis in the US cell-derived spheroids. Conclusions: Our studies demonstrate that Entinostat, in combination with Tazemetostat proves significantly superior to either single treatment. Dual targeting EZH2 and HDACs may provide a promising treatment option for this aggressive cancer disease.

Sunitinib attenuates reactive MDSCs enhancing anti-tumor immunity in HNSCC

Enhancer of zeste homolog 2 (EZH2) is implicated in promoting HNSCC malignant progression. However, EZH2 inhibitors, when used alone, increase the number of myeloid-derived suppressor cells (MDSCs), which are responsible for enhancing tumor stemness and promoting tumor immune escape. We aimed to determine whether combining tazemetostat (an EZH2 inhibitor) and sunitinib (a MDSC inhibitor) can improve the response rate to an immune-checkpoint-blocking (ICB) therapy. We evaluated the efficacy of the above treatment strategies by bioinformatics analysis and animal experiments. EZH2 overexpression and abundant MDSCs in patients with HNSCC are associated with tumor progression. Tazemetostat treatment alone had limited inhibitory effect on HNSCC progression in the mouse models, accompanied by a surge in the number of MDSCs in the tumor microenvironment. Conversely, the combined use of tazemetostat and sunitinib reduced the number of MDSCs and regulatory T cell populations, promoting intratumoral infiltration of T cells and inhibiting of T cell exhausting, regulating of wnt/β-catenin signaling pathway and tumor stemness, promoting the intratumoral PD-L1 expression and improved the response rate to anti-PD-1 therapy. The combined use of EZH2 and MDSC inhibitors effectively reverses HNSCC-specific immunotherapeutic resistance and is a promising strategy for overcoming resistance to ICB therapy.

Phase I study of the bifunctional anti-PD-L1/TGF-βRII agent SHR-1701 combined with SHR2554, an EZH2 inhibitor, in patients with previously treated advanced lymphoma and solid tumors.

2507 Background: Despite the remarkable successes of cancer immunotherapies, the majority of patients (pts) experience only partial response and most pts will progress. Enhancer of zeste homolog 2 (EZH2) as an epigenetic target for cancer therapy has been shown to enhanced antigen presentation and synergize with immunotherapy in preclinical studies. This study investigates the safety and efficacy of SHR-1701 (anti-PD-L1/TGF-βRII) plus SHR2554 (EZH2i) in pts with pretreated advanced lymphoma and solid tumors. Methods: This open-label, single-arm, phase 1 study enrolled pts with pretreated advanced lymphoma and solid tumors. Escalating doses of SHR2554 (300–350mg bid) plus fixed-dose SHR-1701 (30mg/kg q3w) were evaluated in the dose-escalation phase. SHR2554 at the recommended phase 2 dose (RP2D) plus SHR-1701 were administered in the dose-expansion phase. The primary end point was determination of the RP2D of SHR2554 plus SHR-1701. Secondary end points included safety and preliminary activity. Results: By January 6, 2023, 33 patients were enrolled (dose-escalation: n=7; dose-expansion, n=26). No dose-limiting toxicities were observed, and RP2D was established as SHR2554 350mg bid plus SHR-1701 30mg/kg q3w. Treatment-related adverse events (TRAEs) occurred in 13 of 32 patients (40.6%), mainly including platelet count decreased (21.9%), anemia (15.6%) and vomiting (15.6%). The most common Grade ≥3 TRAEs were anemia (3.1%) and fever (3.1%). In the efficacy-evaluable population (n = 26), objective response rate (ORR) was 57.7%, including 2 complete response (CR) achieved by patients with Metastatic Renal Cell Carcinoma and classical Hodgkin lymphoma (cHL). 16 patients with relapsed/refractory classical Hodgkin lymphoma (cHL) after a median of 10 prior lines of therapy have been enrolled. 16 (100%) cHL pts had received prior anti-PD1/PD-L1 antibody therapy, and 15 ( 93.8%) prior epigenetic therapies, such as decitabine or chidamide. Out of 14 cHL-evaluable patients, ORR was 100% and the CR rate was 7.1%. Conclusions: SHR-1701 combined with SHR2554 showed acceptable safety profile and encouraging antitumor activity in immunotherapy-pretreated cHL, establishing the foundation for further exploration. Clinical trial information: NCT04407741 .

USP7 prevents recurrent spontaneous abortion by targeting EZH2 to regulate trophoblast proliferation, apoptosis, migration and invasion through the Wnt/β-catenin pathway†.

Trophoblasts are significant components of the placenta and play crucial roles in maternal-fetal crosstalk. Adequate trophoblast migration and invasion are essential for embryo implantation and a healthy pregnancy. Ubiquitin-specific protease 7 (USP7), a member of the deubiquitinating enzyme family, regulates the processes of migration and invasion in multiple tumor cells. However, the effects of USP7 on trophoblasts and its possible mechanism in the development of recurrent spontaneous abortion (RSA) are still unclear. In this study, we analyzed the expression of USP7 in villous tissues obtained from RSA patients and healthy controls, and then GNE-6776 (a USP7-specific inhibitor) and USP7 siRNA were used in a trophoblast cell line, HTR-8/SVneo, to further assess the effect of USP7 on the biological function of trophoblasts. Our results provide convincing evidence that USP7 is downregulated in the placental villous tissues of RSA patients. USP7 was found to have a crucial role in the proliferation, apoptosis, migration, invasion and epithelial-mesenchymal transition (EMT) process of trophoblast cells. Further experiments revealed that USP7 directly interacted with enhancer of zeste homolog 2 (EZH2) and regulated the Wnt/β-catenin signaling pathway in trophoblasts. Taken together, these findings indicate the vital role of USP7 in regulating trophoblast proliferation, migration and invasion, thus affecting the pathogenesis of RSA, providing new insights into the important role of USP7 in the maternal-fetal interface.

Discovery of a New-Generation S-Adenosylmethionine-Noncompetitive Covalent Inhibitor Targeting the Lysine Methyltransferase Enhancer of Zeste Homologue 2.

The first-generation enhancer of zeste homologue 2 (EZH2) inhibitors suffer from several limitations, such as high dosage, cofactor S-adenosylmethionine (SAM) competition, and acquired drug resistance. Development of covalent EZH2 inhibitors that are noncompetitive with cofactor SAM offers an opportunity to overcome these disadvantages. The structure-based design of compound 16 (BBDDL2059) as a highly potent and selective covalent inhibitor of EZH2 is presented in this context. 16 inhibits EZH2 enzymatic activity at sub-nanomolar concentrations and achieves low nanomolar potencies in cell growth inhibition. The kinetic assay revealed that 16 is noncompetitive with the cofactor SAM, providing the basis for its superior activity over noncovalent and positive controls by reducing competition with cofactor SAM and offering a preliminary proof for its covalent inhibition nature. Mass spectrometric analysis and washout experiments firmly establish its covalent inhibition mechanism. This study demonstrates that covalent inhibition of EZH2 can offer a new opportunity for the development of promising new-generation drug candidates.

Pharmacological inhibition of EZH2 using a covalent inhibitor suppresses human ovarian cancer cell migration and invasion.

Metastasis is the cause of poor prognosis in ovarian cancer (OC). Enhancer of Zeste homolog 2 (EZH2), a histone-lysine N-methyltransferase enzyme, promotes OC cell migration and invasion by regulating the expression of tissue inhibitor of metalloproteinase-2 (TIMP2) and matrix metalloproteinases-9 (MMP9). Hence, we speculated that EZH2-targeting therapy might suppress OC migration and invasion. In this study, the expression of EZH2, TIMP2, and MMP9 in OC tissues and cell lines was analyzed using The Cancer Genome Atlas (TCGA) database and western blotting, respectively. The effects of SKLB-03220, an EZH2 covalent inhibitor, on OC cell migration and invasion were investigated using wound-healing assays, Transwell assays, and immunohistochemistry. TCGA database analysis confirmed that the EZH2 and MMP9 mRNA expression was significantly higher in OC tissues, whereas TIMP2 expression was significantly lower than that in normal ovarian tissues. Moreover, EZH2 negatively correlated with TIMP2 and positively correlated with MMP9 expression. In addition to the anti-tumor activity of SKLB-03220 in a PA-1 xenograft model, immunohistochemistry results showed that SKLB-03220 markedly increased the expression of TIMP2 and decreased the expression of MMP9. Additionally, wound-healing and Transwell assays showed that SKLB-03220 significantly inhibited the migration and invasion of both A2780 and PA-1 cells in a concentration-dependent manner. SKLB-03220 inhibited H3K27me3 and MMP9 expression and increased TIMP2 expression in PA-1 cells. Taken together, these results indicate that the EZH2 covalent inhibitor SKLB-03220 inhibits metastasis of OC cells by upregulating TIMP2 and downregulating MMP9, and could thus serve as a therapeutic agent for OC.

The roles of EZH2 in cancer and its inhibitors.

The enhancer of zeste homolog 2 (EZH2) is encoded by the Enhancer of zeste 2 polycomb repressive complex 2 subunit gene. EZH2 is involved in the cell cycle, DNA damage repair, cell differentiation, autophagy, apoptosis, and immunological modulation. The main function of EZH2 is to catalyze the methylation of H3 histone of H3K27Me3, which inhibits the transcription of target genes, such as tumor suppressor genes. EZH2 also forms complexes with transcriptions factors or directly binds to the promoters of target genes, leading to regulate gene transcriptions. EZH2 has been as a prominent target for cancer therapy and a growing number of potential targeting medicines have been developed. This review summarized the mechanisms that EZH2 regulates gene transcription and the interactions between EZH2 and important intracellular signaling molecules (Wnt, Notch, MEK, Akt) and as well the clinical applications of EZH2-targeted drugs.

EZH2 inhibition remodels the inflammatory senescence-associated secretory phenotype to potentiate pancreatic cancer immune surveillance.

Immunotherapies that produce durable responses in some malignancies have failed in pancreatic ductal adenocarcinoma (PDAC) due to rampant immune suppression and poor tumor immunogenicity. We and others have demonstrated that induction of the senescence-associated secretory phenotype (SASP) can be an effective approach to activate anti-tumor natural killer (NK) cell and T cell immunity. In the present study, we found that the pancreas tumor microenvironment suppresses NK cell and T cell surveillance after therapy-induced senescence through enhancer of zeste homolog 2 (EZH2)-mediated epigenetic repression of proinflammatory SASP genes. EZH2 blockade stimulated production of SASP chemokines CCL2 and CXCL9/10, leading to enhanced NK cell and T cell infiltration and PDAC eradication in mouse models. EZH2 activity was also associated with suppression of chemokine signaling and cytotoxic lymphocytes and reduced survival in patients with PDAC. These results demonstrate that EZH2 represses the proinflammatory SASP and that EZH2 inhibition combined with senescence-inducing therapy could be a powerful means to achieve immune-mediated tumor control in PDAC.

Alteration of the tumor microenvironment by pharmacological inhibition of EZH2 in hepatocellular carcinoma.

Enhancer of zeste homolog 2 (EZH2), a core component of polycomb repressive component 2 is overexpressed in a variety of cancers and recognized as a therapeutic target molecule. However, EZH2 possesses immunomodulatory functions in the tumor microenvironment (TME). The impact of EZH2 on TME of hepatocellular carcinoma (HCC) using immunocompetent mouse model was evaluated in the present study. UNC1999, an EZH2 inhibitor, impaired growth of the murine HCC cells (H22 cells) and induced apoptosis in a dose-dependent manner. Although UNC1999 significantly inhibited the growth of H22 cells-derived and Hepa1-6 cells-derived tumors in nonobese diabetic/severe combined immunodeficiency mice, its antitumor effect was diminished in allogenic BALB/c and C57BL/6 mice. Flow cytometric analyses of TME cells in BALB/c mice demonstrated a significant decrease in the number of interferon‑γ+ CD8+ T cells and regulatory T cells and a significant increase in the number of myeloid-derived suppressor cells (MDSCs). Administration of Gr-1 neutralizing antibody concomitant with UNC1999 restored antitumor effect accompanied by an increase in the number of CD8+ T cells followed by a decrease in the number of MDSCs. Chemokine antibody array demonstrated an enhanced expression of chemokines responsible for MDSCs recruitment such as C5a, CCL8, and CCL9. In conclusion, the study results demonstrated that EZH2 inhibitor contributed to attenuation of tumor immunity caused by TME arrangement. Combination therapy with EZH2 inhibitors and agents that reduce MDSCs might represent a novel therapeutic strategy for HCC.

Targeting B7‑H3 through EZH2 inhibition in MYC‑positive Group 3 medulloblastoma.

The most aggressive subtype of medulloblastoma (MB), Group 3, is characterized by MYC amplifications. However, targeting MYC has proven unsuccessful, and there remains a lack of therapeutic targets for treating MB. Studies have shown that the B7 homolog 3 (B7‑H3) promotes cell proliferation and tumor cell invasion in a variety of cancers. Similarly, it was recently revealed that B7‑H3 promotes angiogenesis in Group 3 MB and likely facilitates MB metastasis through exosome biogenesis. While therapies targeting B7‑H3 remain in the early stages of development, targeting upstream regulators of B7‑H3 expression may be more effective for halting MB progression. Notably, MYC and the enhancer of zeste homolog 2 (EZH2) are known to regulate B7‑H3 expression, and a previous study by the authors suggested that B7‑H3 amplifications present in MB are likely the result of EZH2‑MYC mediated activities. In the present study, it was reported that overexpression of EZH2 is associated with lower overall survival in Group 3 MB patients. It was also revealed that inhibition of EZH2 significantly reduces B7‑H3 and MYC transcript levels and upregulates miR‑29a, indicating that EZH2 post‑transcriptionally regulates B7‑H3 expression in Group 3 MB cells. Pharmacological inhibition of EZH2 using EPZ005687 attenuated MB cell viability and reduced the expression of B7‑H3. Similarly, pharmacological inhibition and knockdown of EZH2 led to the downregulation of MYC, B7‑H3, and H3K27me3. Further, EZH2 silencing induced apoptosis and reduced colony‑forming ability in MB cells, whereas EZH2 inhibition in MYC‑amplified C17.2 neural stem cells induced G2/M phase arrest while downregulating B7‑H3 expression. Collectively, the current study positions EZH2 as a viable target for the future development of MB treatments and that targeting EZH2 in combination with B7‑H3 immunotherapy may be an effective treatment for halting MB progression.

Combining EZH2 and HDAC inhibitors to target castration-resistant prostate cancers.

Development of resistance in castration-resistant prostate cancer (CRPC) involves epigenetic pathways. A new study in PLOS Biology demonstrates that combined therapy targeting enhancer of zeste homolog 2 (EZH2) and histone deacetylases (HDACs) may sensitize CRPC to both epigenetic and standard therapies.

Meta-analysis of Genetic polymorphism of Enhancer of Zeste Homolog2 gene in cancer susceptibility.

The alteration in the expression of enhancer of zeste homolog-2 (EZH2) gene is very well known in the progression, severity, and aggressiveness of cancer. Hence, it is important to study the genomic variation of the EZH2 gene. Previously, many association-based studies investigated the association between rs2302427C>G and cancer susceptibility. However, the result had been inconsistent. Therefore, our meta-analysis aimed to identify the association between EZH2 rs2302427 and cancer risk. A systematic literature search was done for databases PubMed, Google Scholar, Science Direct, and Cochrane library up to September 2020 and statistical analysis was performed by RevMan v 5.3. A total of six studies comprised 1876 cases and 2555 controls were included in the current meta-analysis. The pooled analysis showed that overall, there is significant association of rs2302427 C>G change with reduced cancer risk (odds ratio = 0.60, 95% confidence interval [0.35-1.03], P = 0.07) but non-significantly. Further, the subgroup analysis also revealed that there is no significant difference between the Asian and European population, and both exhibit the protective nature of rs2302427 with cancer. The present meta-analysis indicated that EZH2 rs2302427 has an association with cancer in reducing the risk but for the Indian population studies are required as the Indian population comprises various sub-population genetically isolated for long.

EZH2 as a prognostic-related biomarker in lung adenocarcinoma correlating with cell cycle and immune infiltrates

BackgroundsIt has been observed that high levels of enhancer of zeste homolog 2 (EZH2) expression are associated with unsatisfactory prognoses and can be found in a wide range of malignancies. However, the effects of EZH2 on Lung Adenocarcinoma (LUAD) remain elusive. Through the integration of bioinformatic analyses, the present paper sought to ascertain the effects of EZH2 in LUAD.MethodsThe TIMER and UALCAN databases were applied to analyze mRNA and protein expression data for EZH2 in LUAD. The result of immunohistochemistry was obtained from the HPA database, and the survival curve was drawn according to the library provided by the HPA database. The LinkedOmics database was utilized to investigate the co-expressed genes and signal transduction pathways with EZH2. Up- and down-regulated genes from The Linked Omics database were introduced to the CMap database to predict potential drug targets for LUAD using the CMap database. The association between EZH2 and cancer-infiltrating immunocytes was studied through TIMER and TISIDB. In addition, this paper explores the relationship between EZH2 mRNA expression and NSCLC OS using the Kaplan–Meier plotter database to further validate and complement the research. Furthermore, the correlation between EZH2 expression and EGFR genes, KRAS genes, BRAF genes, and smoking from the Cancer Genome Atlas (TCGA) database is analyzed.ResultsIn contrast to paracancer specimens, the mRNA and protein levels of EZH2 were higher in LUAD tissues. Significantly, high levels of EZH2 were associated with unsatisfactory prognoses in LUAD patients. Additionally, the coexpressed genes of EZH2 were predominantly associated with numerous cell growth-associated pathways, including the cell cycle, DNA replication, RNA transport, and the p53 signaling pathway, according to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways. The results of TCGA database revealed that the expression of EZH2 was lower in normal tissues than in lung cancer tissues (p < 0.05). Smoking was associated with elevated EZH2 expression (p < 0.001). EZH2 was highly expressed in lung cancers with positive KRAS expression, and the correlation was significant in lung adenocarcinoma (r = 0.3129, p < 0.001). CMap was applied to determine the top 15 positively correlated drugs/molecules and the top 15 negatively correlated drugs/molecules. MK-1775, MK-5108, fenbendazole, albendazole, BAY-K8644, evodiamine, purvalanol-a, mycophenolic-acid, PHA-793887, and cyclopamine are potential drugs for patients with lung adenocarcinoma and high EZH2 expression.ConclusionsHighly expressed EZH2 is a predictor of a suboptimal prognosis in LUAD and may serve as a prognostic marker and target gene for LUAD. The underlying cause may be associated with the synergistic effect of KRAS, immune cell infiltration, and metabolic processes.

Dual targeting of aberrant DNA and histone methylation synergistically suppresses tumor cell growth in ATL.

Adult T-cell leukemia/lymphoma (ATL) is a malignancy of mature CD4+ T cells caused by human T-cell lymphotropic virus type 1 (HTLV-1)-induced T-cell transformation. After infection with HTLV-1, it takes several decades for HTLV-1 carriers to develop ATL. The prognosis of ATL remains poor despite several new agents being approved in the last few years. Recently, it has been noted that epigenetic abnormalities, both DNA methylation and trimethylation at histone H3Lys27 (H3K27me3), contribute to ATL leukemogenesis. Here, we investigated the effect of combination treatment with DNA demethylating agents (azacitidine [AZA], decitabine (DAC), and OR-2100 (OR21), which is a silylated derivative of DAC) and inhibitors of enhancer of zeste homolog 2 (EZH2) (EPZ-6438 and DS-3201b), which catalyze trimethylation of H3K27, in ATL. The combination of DAC and OR21 but not AZA with EZH inhibitors exhibited synergistic anti-ATL effects invitro and invivo, concomitant with DNA demethylation and reduction of H3K27me3. The combination induced gene expression reprogramming. Dual-specificity phosphatase 5 (DUSP5), an extracellular signal-regulated kinase (ERK)-specific phosphatase, was identified as a key molecule that mediated the inhibitory effect of combination treatment by inactivating the ERK signaling pathway. DUSP5 was downregulated by DNA methylation and H3K27me3 accumulation in the promoter region in HTLV-1-infected cells from patients with ATL during ATL leukemogenesis. The present results demonstrate that dual targeting of aberrant DNA and histone methylation synergistically suppresses tumor cell growth by restoring DUSP5, and that dual targeting of aberrant DNA and histone methylation is a feasible therapeutic approach for ATL.

Abstract CT105: Preliminary results from the Phase I part of a first-in-human Phase I/II study of HH2853, an EZH1/2 inhibitor, in patients with relapsed/refractory non-Hodgkin lymphomas or advanced solid tumors

Abstract Background: The dysregulation of polycomb repressive complex 2 (PRC2) promotes tumorigenesis and progression. Two therapeutic agents targeting enhancer of zeste homolog (EZH) 2 or EZH1/2, the catalytic subunits of PRC2, have been approved in several cancer types. HH2853 is a novel selective EZH1/2 dual inhibitor, which has demonstrated superior anti-tumor efficacy to tazemetostat (EZH2 inhibitor approved by FDA) in various preclinical models. Methods: This is a first-in-human, open-label, multi-center, phase (Ph) I/II study of HH2853 in patients (pts) with relapsed/refractory (r/r) non-Hodgkin lymphomas (NHLs) or advanced solid tumors. HH2853 was administered orally twice daily (BID) on a continuous 28-day treatment cycle. Ph I consist of two parts: dose escalation part adopting accelerated titration followed by a Bayesian optimal interval design and dose extension part. Dose limiting toxicity (DLT) was evaluated during the 1st cycle in dose escalation. Safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD) and preliminary anti-tumor activity of HH2853 were explored in this Ph I study. Results: As of Oct 19, 2022, a total of 57 pts, including 50 pts with solid tumors and 7 pts with r/r follicular lymphoma (FL), were enrolled from 12 sites in China and the US. Twenty-five (43.9%) pts received ≥3 lines of prior systemic therapies. Six dose levels(50 mg, 100 mg, 200 mg, 400 mg, 600 mg and 800 mg)were evaluated. DLTs were observed in 2 of 8 DLT evaluable pts at 800 mg: one grade 3 platelet count decreased and one grade 3 diarrhea. A conclusive maximal tolerated dose was not reached. The most common treatment-related adverse events (TRAE) were diarrhea (45.6%), blood bilirubin increased (35.1%), white blood cell count (WBC) decreased (26.3%), platelet count decreased (26.3%), rash (24.6%) and anemia (22.8%). The most common ≥grade 3 TRAEs included anemia (8.8%), platelet count decreased (7.0%), WBC decreased (5.3%) and diarrhea (5.3%). TRAEs leading to dose interruption or reduction were reported in 17.5% and 8.8% pts respectively. No TRAE led to dose discontinuation or death. PK data indicated dose-related increase in exposure from 50 to 600 mg. PD data showed a significant inhibition (maximum reached &amp;gt;90%) of H3K27me3 in granulocytes and monocytes at 400-800 mg. Tumor responses were observed in 7 pts from 200 to 800 mg in multiple tumor types, including complete response in 1 patient with FL, partial response in 3 pts with epithelioid sarcoma, 2 pts with FL and 1 patient with malignant rhabdoid tumor of pancreas. Conclusions: This first-in-human study of HH2853 showed a manageable safety profile and promising anti-tumor activity in multiple tumor types, supporting further exploration in NHLs and solid tumors after recommended Ph II dose is determined.Clinical trial information: NCT04390737 Citation Format: Zhengfu Fan, Jin Wang, Meiyu Fang, Johnston Patrick, Tun Han, David Sommerhalder, Jifang Gong, Jilong Yang, Yun Yang, Javier Munoz, Yuqin Song, Zhiming Li, Xian'an Li, Qiuying Ma, Jinming Han, Lin Shen, Jun Zhu. Preliminary results from the Phase I part of a first-in-human Phase I/II study of HH2853, an EZH1/2 inhibitor, in patients with relapsed/refractory non-Hodgkin lymphomas or advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT105.

EZH2 controls epicardial cell migration during heart development.

Enhancer of zeste homolog 2 (EZH2) is an important transcriptional regulator in development that catalyzes H3K27me3. The role of EZH2 in epicardial development is still unknown. In this study, we show that EZH2 is expressed in epicardial cells during both human and mouse heart development. Ezh2 epicardial deletion resulted in impaired epicardial cell migration, myocardial hypoplasia, and defective coronary plexus development, leading to embryonic lethality. By using RNA sequencing, we identified that EZH2 controls the transcription of tissue inhibitor of metalloproteinase 3 (TIMP3) in epicardial cells during heart development. Loss-of-function studies revealed that EZH2 promotes epicardial cell migration by suppressing TIMP3 expression. We also found that epicardial Ezh2 deficiency-induced TIMP3 up-regulation leads to extracellular matrix reconstruction in the embryonic myocardium by mass spectrometry. In conclusion, our results demonstrate that EZH2 is required for epicardial cell migration because it blocks Timp3 transcription, which is vital for heart development. Our study provides new insight into the function of EZH2 in cell migration and epicardial development.

Distinct Regulation of EZH2 and its Repressive H3K27me3 Mark in Polyomavirus-Positive and -Negative Merkel Cell Carcinoma

Merkel cell carcinoma (MCC) is an aggressive skin cancer for which Merkel cell polyomavirus integration and expression of viral oncogenes small T and Large T have been identified as major oncogenic determinants. Recently, a component of the PRC2 complex, the histone methyltransferase enhancer of zeste homolog 2 (EZH2) that induces H3K27 trimethylation as a repressive mark has been proposed as a potential therapeutic target in MCC. Because divergent results have been reported for the levels of EZH2 and trimethylation of lysine 27 on histone 3, we analyzed these factors in a large MCC cohort to identify the molecular determinants of EZH2 activity in MCC and to establish MCC cell lines' sensitivity to EZH2 inhibitors. Immunohistochemical expression of EZH2 was observed in 92% of MCC tumors (156 of 170), with higher expression levels in virus-positive than virus-negative tumors (P= 0.026). For the latter, we showed overexpression of EZHIP, a negative regulator of the PRC2 complex. Invitro, ectopic expression of the large T antigen in fibroblasts led to the induction of EZH2 expression, whereas the knockdown of T antigens in MCC cell lines resulted in decreased EZH2 expression. EZH2 inhibition led to selective cytotoxicity on virus-positive MCC cell lines. This study highlights the distinct mechanisms of EZH2 induction between virus-negative and -positive MCC.

Dual role of enhancer of zeste homolog 2 in the regulation of ultraviolet radiation-induced matrix metalloproteinase-1 and type I procollagen expression in human dermal fibroblasts

Abnormalities in the extracellular matrix (ECM) caused by ultraviolet (UV) radiation are mediated by epigenetic mechanisms. Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase that is implicated in inflammation, immune regulation, and senescence. However, its role in controlling UV-induced ECM alterations in the skin remains elusive. Here, we investigated the role of EZH2 in UV-induced expression of matrix metalloproteinase (MMP)-1 and type I procollagen. We found that UV induced EZH2 expression in human skin in vivo and in human dermal fibroblasts (HDFs). EZH2 knockdown reduced the expression and promoter activity of MMP-1 and increased those of type I procollagen, whereas EZH2 overexpression had the opposite effects. Mechanistically, EZH2 increased NF-κB activity, and p65 and p50 expression and promoter activity. Intriguingly, chromatin immunoprecipitation assays revealed that the EZH2/p65/p50 complex was recruited and bound to the MMP-1 promoter after UV irradiation, independent of its histone methyltransferase activity. In contrast, EZH2-induced DNA methyltransferase 1 (DNMT1) formed a complex with EZH2 and enhanced the enrichment of H3K27me3 on the COL1A2 promoter following UV irradiation. These findings indicate that EZH2 plays a dual role in regulating MMP-1 and type I procollagen expression and improve our understanding of how this epigenetic mechanism contributes to UV-induced skin responses and photoaging. This study shows that inhibiting EZH2 is a potential anti-aging strategy for preventing UV-induced skin aging by reducing MMP-1 expression and inducing type I procollagen expression.

Enhancer of zeste homolog 2 promotes renal fibrosis after acute kidney injury by inducing epithelial-mesenchymal transition and activation of M2 macrophage polarization

Long-term follow-up data indicates that 1/4 patients with acute kidney injury (AKI) will develop to chronic kidney disease (CKD). Our previous studies have demonstrated that enhancer of zeste homolog 2 (EZH2) played an important role in AKI and CKD. However, the role and mechanisms of EZH2 in AKI-to-CKD transition are still unclear. Here, we demonstrated EZH2 and H3K27me3 highly upregulated in kidney from patients with ANCA-associated glomerulonephritis, and expressed positively with fibrotic lesion and negatively with renal function. Conditional EZH2 deletion or pharmacological inhibition with 3-DZNeP significantly improved renal function and attenuated pathological lesion in ischemia/reperfusion (I/R) or folic acid (FA) mice models (two models of AKI-to-CKD transition). Mechanistically, we used CUT & Tag technology to verify that EZH2 binding to the PTEN promoter and regulating its transcription, thus regulating its downstream signaling pathways. Genetic or pharmacological depletion of EZH2 upregulated PTEN expression and suppressed the phosphorylation of EGFR and its downstream signaling ERK1/2 and STAT3, consequently alleviating the partial epithelial-mesenchymal transition (EMT), G2/M arrest, and the aberrant secretion of profibrogenic and proinflammatory factors in vivo and vitro experiments. In addition, EZH2 promoted the EMT program induced loss of renal tubular epithelial cell transporters (OAT1, ATPase, and AQP1), and blockade of EZH2 prevented it. We further co-cultured macrophages with the medium of human renal tubular epithelial cells treated with H2O2 and found macrophages transferred to M2 phenotype, and EZH2 could regulate M2 macrophage polarization through STAT6 and PI3K/AKT pathways. These results were further verified in two mice models. Thus, targeted inhibition of EZH2 might be a novel therapy for ameliorating renal fibrosis after acute kidney injury by counteracting partial EMT and blockade of M2 macrophage polarization.