H3K27me3 Research Articles

EZH2 Promotes Cholangiocarcinoma Development and Progression through Histone Methylation and microRNA-Mediated Down-Regulation of Tumor Suppressor Genes

Cholangiocarcinoma (CCA) is a highly malignant cancer of the biliary tree. Although studies have implicated enhancer of Zeste homolog 2 (EZH2) in CCA growth, the role of EZH2 in CCA development has not been investigated, and the mechanism for EZH2-regulated gene expression in CCA remains to be further defined. The current study used a mouse model of CCA induced by hydrodynamic tail vein injection of Notch1 intracellular domain and myristoylated-AKT plasmids. Mice with liver-specific EZH2 knockout displayed reduced CCA development. In a xenograft model, EZH2 knockdown significantly decreased CCA progression. Administration of the EZH2 inhibitor GSK126 decreased CCA tumor burden in mice. Accordingly, EZH2 depletion or inhibition reduced the growth and colony formation capability of CCA cells. Analysis of high-throughput data identified a set of 12 tumor-inhibiting genes as targets of EZH2 in CCA. The experimental results suggest that EZH2 may down-regulate these tumor-inhibiting genes through methylation of lysine 27 on histone H3 (H3K27) in the gene louses and through regulation of specific miRNAs. High mobility group box 1 was shown to facilitate the methyltransferase activity of EZH2, which is implicated in the regulation of CCA cell growth. The study shows that EZH2 promotes CCA development and progression through a complicated regulatory network involving tumor-inhibiting genes, miRNAs, and high mobility group box 1, which support targeting EZH2 as a potentially effective strategy for CCA treatment.

Loss of SUV420H2-Dependent Chromatin Compaction Drives Right-Sided Colon Cancer Progression

Epigenetic processes regulating gene expression contribute markedly to epithelial cell plasticity in colorectal carcinogenesis. The lysine methyltransferase SUV420H2 comprises an important regulator of epithelial plasticity and is primarily responsible for trimethylation of H4K20 (H4K20me3). Loss of H4K20me3 has been suggested as a hallmark of human cancer due to its interaction with DNMT1. However, the role of Suv4-20h2 in colorectal cancer is unknown. We examined the alterations in histone modifications in patient-derived colorectal cancer organoids. Patient-derived colorectal cancer organoids and mouse intestinal organoids were genetically manipulated for functional studies in patient-derived xenograft and orthotopic transplantation. Gene expression profiling, micrococcal nuclease assay, and chromatin immunoprecipitation were performed to understand epigenetic regulation of chromatin states and gene expression in patient-derived and mouse intestinal organoids. We found that reduced H4K20me3 levels occurred predominantly in right-sided patient-derived colorectal cancer organoids, which were associated with increased chromatin accessibility. Re-compaction of chromatin by methylstat, a histone demethylase inhibitor, resulted in reduced growth selectively in subcutaneously grown tumors derived from right-sided cancers. Using mouse intestinal organoids, we confirmed that Suv4-20h2-mediated H4K20me3 is required for maintaining heterochromatin compaction and to prevent R-loop formation. Cross-species comparison of Suv4-20h2-depleted murine organoids with right-sided colorectal cancer organoids revealed a large overlap of gene signatures involved in chromatin silencing, DNA methylation, and stemness/Wnt signaling. Loss of Suv4-20h2-mediated H4K20me3 drives right-sided colorectal tumorigenesis through an epigenetically controlled mechanism of chromatin compaction. Our findings unravel a conceptually novel approach for subtype-specific therapy of this aggressive form of colorectal cancer.

Genome-Wide CRISPR-Cas9 Screen Identifies KDM6A As a Modulator of Daratumumab Sensitivity in Multiple Myeloma

Daratumumab (Dara) is the first-in-class monoclonal antibody targeting CD38, which triggers both direct and immune-mediated cytotoxicity of multiple myeloma (MM) cells in the bone marrow (BM) milieu. Importantly, Dara combination therapies have a high frequency and extent of response in both relapsed/refractory and newly diagnosed MM. However, relapse is commonly observed due to the development of Dara resistance. As an immune-targeting drug, the anti-tumor activity of Dara is closely associated with the expression of CD38, with nonresponders having a lower expression of CD38. To identify novel regulators mediating CD38 expression and Dara sensitivity in MM cells, we performed a genome-wide CRISPR-Cas9 knockout (KO) screen in vitro. We identified 46 different sgRNAs that were positively correlated with MM cytotoxicity triggered by Dara and primary NK cells. Using the cells with the lowest expression of CD38 (bottom 5%), we performed a second round of screening and found that KDM6A was the top-ranked gene out of the set of overlapping genes from these two screenings. We confirmed that CD38 expression was significantly decreased (>2 times, p<0.01) by KDM6A KO and that re-introducing KDM6A into KDM6A KO cells restored the expression level of CD38 and Dara-induced cytotoxicity. KDM6A encodes lysine-specific demethylase 6A, which demethylates lysine 27 of histone H3 (H3K27) to promote gene expression. To confirm that CD38 expression was altered by the methylation status of H3K27, we compared the CD38 promoter area between KDM6A KO and control cells using ChIP-seq and ChIP qPCR and found that the H3K27me3 level was higher in KDM6A KO cells. H3K27 is methylated by EZH2, and we found that EZH2 inhibitor treatment significantly decreased the H3K27me3 level of the CD38 promoter area. Moreover, EZH2 inhibitor treatment upregulated both CD38 mRNA and protein expression in KDM6A KO cells. Importantly, EZH2 inhibitor also restored the sensitivity of KDM6A KO cells to Dara-mediated NK cell cytotoxicity in vitro and in xenograft mouse model. We next re-introduced CD38 into KDM6A KO cells and found that CD38 overexpression only partially restored the sensitivity of KDM6A KO cells to Dara treatment. This suggested that besides CD38 downregulation, there are other mechanisms underlying Dara resistance induced by KDM6A KO. We performed RNA-seq in KDM6A KO cells and found that CD48, an NK-activating ligand, was downregulated. Overexpression of CD48 in KDM6A KO cells significantly increased secretion of Granzyme B and Perforin, restoring the activity of NK cells and re-sensitizing KDM6A KO cells to Dara treatment. These data suggest that KDM6A not only regulates CD38 expression, but also NK cell activity by CD48 regulation, thereby modulating the efficacy of Dara. Taken together, our studies demonstrate that KDM6A regulates CD38 and CD48 expression in MM. Moreover, we validate that combination treatment with FDA-approved EZH2 inhibitor and Dara can overcome Dara resistance in preclinical MM models, providing the rationale for combination clinical trials to overcome Dara resistance and improve patient outcome.

An Open-Label, Multicenter,Single-Arm, Phase I Study to Evaluate the Safety, Pharmacokinetics, Pharmacodynamics and Efficacy of XNW5004 in Patients with Relapsed or Refractory Hematologic Malignancies

Background: The abnormal regulation and overexpression of Enhancer of zeste homolog 2 (EZH2) was tested in advanced tumor. EZH2 inhibits the transcription of target genes by trimethylation of lysine 27 on histone H3 (H3K27me3), and its activity-enhancing mutation and abnormal expression are the decisive factor for abnormal transcription in many tumors. Besides, EZH2 significantly correlated with tumor malignancy and poor prognosis. XNW5004 is a class of small-molecule EZH2 inhibitor. Preclinical shows that XNW5004 has good safety and anti-tumor efficacy. Methods: This Chinese, open-label, single-arm, multicenter, phase I study is evaluating the safety, tolerability, Pharmacokinetics (PK), Pharmacodynamics (PD), and efficacy of XNW5004 in adult subjects with pathologically confirmed relapsed/refractory non-Hodgkin lymphoma (NHL). Eligibility criteria included an Eastern Cooperative Oncology Group (ECOG) performance score of 0-1, life expectancy ≥12 weeks, and adequate bone marrow, renal, and liver function. The study uses an accelerated titration and traditional 3+3 dose-escalated design, with a cycle of 28 days. This study reports data on pts with dose-escalated phase. Results: In the dose-escalated phase, as July 15, 2022 , a total of 15 pts were enrolled and treated with 5 dose groups of XNW5004 (from 100 mg to 1600 mg BID). The median age was 58 years (range, 47-71). Pts received a median of 3 (range, 2-7) prior lines of treatment and diagnoses of diffuse large B-cell lymphoma (DLBCL, n = 3), follicular lymphoma (FL, n = 6), mantle cell lymphoma (MCL, n = 1), marginal zone lymphoma (MZL, n = 2), small lymphocytic lymphoma (SLL, n = 1), angioimmunoblastic T-cell lymphoma (AITL, n = 1), and Waldenström macroglobulinemia (WM, n = 1). XNW5004 had a favorable safety and tolerability, and no dose-limiting toxicity (DLT) was observed at doses up to 1600 mg BID. Grade 3 treatment-related adverse events (TRAEs) occurred in 4 (26.7%) pts, including white blood cell decreased, neutrophil count decreased, lymphocyte count decreased, and platelet count decreased. No grade ≥4 adverse events (AEs) and serious TRAEs occurred. Any grade TRAEs in ≥20% of pts included nausea (33.3%), diarrhea (33.3%), alanine aminotransferase increased (26.7%), neutrophil count decreased (26.7%), white blood cell decreased (26.7%), hypertriglyceridemia (26.7%), aspartate aminotransferase increased (20%), vomiting (20%), blood lactate dehydrogenase increased (20%), blood bilirubin increased (20%), pruritus (20%), anemia (20%). With a median follow-up of 4 (range, 2-10) cycles, the objective response rate (ORR) was 33.3%, and the disease control rate (DCR) was 78% in the 9 evaluable pts. The median progression-free-survival (mPFS) and median duration of response (mDOR) were not reached. Among pts with EZH2 wild-type (WT) FL (n=5) , the ORR was 40%, the DCR was 100%, the mPFS and mDOR were not reached. In all pts with stable disease (SD), the lesions continued to shrink. One patient (Figure 2) with DLBCL (non-GCB) received 2 cycles of XNW5004, the lesions were reduced by 59.8%; and after 4 cycles of treatment, the lesions were reduced by more than 70%. One patient (Figure 3) with FL (EZH2 WT) received 2 cycles of XNW5004, the subcutaneous lesions and axillary lymph nodes were significantly reduced, PET-CT showed that uptake was strongly reduced, and the Deauville score (DS) was 5 and 3, respectively. The efficacy evaluation was partial response (PR). After 4 cycles of treatment , PET-CT showed that the metabolism of subcutaneous lesions and axillary lymph nodes continued to decrease, the DS was 2 and 1, respectively, and the efficacy evaluation was complete response (CR). In addition, the PK of XNW5004 was well. Conclusions: XNW5004 was safety, well tolerated, and efficacy in pts with different types of lymphomas despite high disease burden, with encouraging preliminary anti-lymphoma activity observed. These data support ongoing dose-escalated phase , with multiple cohort expansions at multiple doses. Internal Study identifier: XNW5004-I/II-01. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

EXTH-29. RADIOSENSITIZATION BY BRD4 INHIBITION IN DIFFUSE INTRINSIC PONTINE GLIOMA

Abstract Diffuse intrinsic pontine glioma (DIPG), the most frequent brainstem tumor in pediatrics, is one of the devastating childhood cancers, and virtually all patients die within two years after diagnosis. Since these tumors occur in the brainstem which is vital area, there are no surgical options for providing relief to patients, and conventional chemotherapy. Radiation therapy (RT) remains the only effective treatment. Radiosensitization is a mean to improve the therapeutic balance between efficacy and toxicity of RT, but not used in clinically. Here, we found BRD4 inhibitor as a radiosensitizer from drug screening. DIPG shows increased H3 K27 acetylation (H3K27ac), which binds to BET bromodomain protein 4 (BRD4) and they are strongly associated with active transcription. We tested two BRD4 inhibiter (BRD4i : AZD5153 and JQ-1) and genetic BRD4 depletion enhances radiation-induced DNA damage, making it a potential radiosensitizer in the treatment of DIPG. We evaluated the effects of BRD4i on genes expression using RNA sequence, inhibited significantly DNA-repair proteins such as BRCA1 and RAD51. CUT-RUN qPCR showed decreased H3K27ac at BRCA1 and RAD51. Combination with BRD4i and RT inhibited cell viability significantly in clonogenic survival assay, apoptosis assay, and showed cell cycle arrest. Radiation-induced DNA double-strand break (DSB) repair was prolonged high level of rH2AHX and 53BP1 with BRD4i because of inhibited DNA-repair. In vivo studies revealed increased survival of animals treated with combination therapy of RT and BRD4i in compared to either monotherapy. Together, these results highlight BRD4i as a potential radiosensitizer and provide a rationale for developing combination therapy with radiation in the treatment of DIPG.

PATH-10. A CASE OF ADULT THALAMIC DIFFUSE MIDLINE GLIOMA, H3 K27-ALTERED WITH AN IMPRESSIVE RESPONSE TO RADIOTHERAPY AND CONCOMITANT PLUS ADJUVANT TEMOZOLOMIDE

Abstract Diffuse midline glioma (DMG), H3 K27-altered is a newly defined diagnosis in the 2021 WHO Classification of Tumors of the Central Nervous System. H3 K27 alterations are associated with a uniformly poor prognosis in children with DMG, but they have been linked to improved survival in adults. Clinical experience in adults with DMG, H3 K27M-altered remains limited. Consequently, there exists a knowledge gap regarding the optimal management and responsiveness to chemoradiation therapy, which translates into the current absence of standard treatment. Here we present the case of an 18-year-old female patient treated for a thalamic DMG, H3 K27-altered at a Canadian tertiary center. The patient first presented with headache, dysarthria, and signs of increased intracranial pressure. Her initial brain magnetic resonance imaging demonstrated a right thalamo-mesencephalic lesion with central nodular enhancement and secondary hydrocephalus. She underwent an endoscopic third ventriculostomy followed by a subtotal resection, and pathology confirmed the presence of a DMG, H3 K27-altered, WHO grade 4. She was treated with combined radiotherapy and concomitant plus adjuvant (12 cycles) temozolomide. The Stupp regimen induced a spectacular response with a reduction in tumor dimensions on T2/FLAIR (20 x 18 mm versus 60 x 51 mm), &amp;gt; 50% decrease in size of the enhancing component, complete resolution of mass effect, and return to functional independence (KPS = 100%). Unfortunately, her disease progressed 16 months after diagnosis and she passed away 8 months later, despite having received 3 cycles of ONC201 through a clinical trial. Her progression-free survival (16 months) was superior to that reported in the literature, which could be related to her young age, subtotal resection status, and thalamic location. Although the addition of chemotherapy to adjuvant radiotherapy has not been shown to improve survival in children with DMG, the Stupp regimen may provide a survival benefit in adults.

ODP438 Canonical and Non-canonical Function of EZH2 in Progesterone Receptor Target Genes in Breast Cancer

Abstract Enhancer of Zeste homolog 2 (EZH2) is a histone methyltransferase which catalyzes the trimethylation of lysine 27 of histone H3 (H3K27me3), an epigenetic mark associated with chromatin compaction and transcriptional repression of target genes. This constitutes its canonical mechanism of action. Several studies have shown that EZH2 is able to activate gene transcription by forming transcriptional complexes through mechanisms that do not involve histone methylation. Indeed, EZH2 can also act in a non-canonical function by regulating transcription independently of its enzymatic activity. We have previously described that progestins induce the interaction between Progesterone Receptor (PR) and EZH2 in breast cancer (BC) cells, which results in the downregulation of tumor suppressor GATA3 and in the increase in cell proliferation. Since EZH2 has been implicated in the progression of several types of cancer, including those of the breast, and our own previous results indicate that EZH2 is required for progestin-induced breast cancer growth, we hypothesized that EZH2 could function as a mediator in the pro-tumorigenic effects of progestins, targeting specific tumor suppressor and differentiating genes to allow ER/PR-positive BC growth. In the present work we found that progestin treatment of T47D cells induced EZH2 mRNA and protein expression, which was abolished by the use of the progestin antagonist RU486 and when PR expression was blocked by siRNAs. We also studied the participation of EZH2 in the regulation of PR-regulated genes TNFalpha (TNFα), cyclin D1 and TIMP2. By ELISA assays, we observed that progestin treatment of T47D cells for 24 h induced TNFα secretion. This effect was abolished when EZH2 expression was inhibited in the presence of EZH2 siRNAs and when EZH2 enzymatic activity was blocked by the use of GSK126. Regarding TIMP2, we observed that EZH2 canonical activity participates in TIMP2 downregulation exerted by progestin treatment. We showed that EZH2 non-canonical function is also involved in progestin modulation of TIMP2 given that blockage of EZH2 activity did not completely revert progestin effect. Finally, we demonstrated that progestin upregulation of cyclin D1 did not require EZH2 activity. Our results prove that EZH2 engagement in PR target genes may involve its canonical or non-canonical function and therefore support further studies on other progestin-regulated cancer genes implicated in tumor growth. Presentation: No date and time listed

Combining EZH2 inhibitors with other therapies for solid tumors: more choices for better effects.

EZH2 is an epigenetic regulator that methylates lysine 27 on histone H3 (H3K27) and is closely related to the development and metastasis of tumors. It often shows gain-of-function mutations in hematological tumors, while it is often overexpressed in solid tumors. EZH2 inhibitors have shown good efficacy in hematological tumors in clinical trials but poor efficacy in solid tumors. Therefore, current research on EZH2 inhibitors has focused on exploring additional combination strategies in solid tumors. Herein we summarize the combinations and mechanisms of EZH2 inhibitors and other therapies, including immunotherapy, targeted therapy, chemotherapy, radiotherapy, hormone therapy and epigenetic therapy, both in clinical trials and preclinical studies, aiming to provide a reference for better antitumor effects.

H3K27me3 in Diffuse Midline Glioma and Epithelial Ovarian Cancer: Opposing Epigenetic Changes Leading to the Same Poor Outcomes.

Histone post-translational modifications modulate gene expression through epigenetic gene regulation. The core histone H3 family members, H3.1, H3.2, and H3.3, play a central role in epigenetics. H3 histones can acquire many post-translational modifications, including the trimethylation of H3K27 (H3K27me3), which represses transcription. Triple methylation of H3K27 is performed by the histone methyltransferase Enhancer of Zeste Homologue 2 (EZH2), a component of the Polycomb Repressive Complex 2. Both global increases and decreases in H3K27me3 have been implicated in a wide range of cancer types. Here, we explore how opposing changes in H3K27me3 contribute to cancer by highlighting its role in two vastly different cancer types; (1) a form of glioma known as diffuse midline glioma H3K27-altered and (2) epithelial ovarian cancer. These two cancers vary widely in the age of onset, sex, associated mutations, and cell and organ type. However, both diffuse midline glioma and ovarian cancer have dysregulation of H3K27 methylation, triggering changes to the cancer cell transcriptome. In diffuse midline glioma, the loss of H3K27 methylation is a primary driving factor in tumorigenesis that promotes glial cell stemness and silences tumor suppressor genes. Conversely, hypermethylation of H3K27 occurs in late-stage epithelial ovarian cancer, which promotes tumor vascularization and tumor cell migration. By using each cancer type as a case study, this review emphasizes the importance of H3K27me3 in cancer while demonstrating that the mechanisms of histone H3 modification and subsequent gene expression changes are not a one-size-fits-all across cancer types.

Characterization of KDM5 lysine demethylase family substrate preference and identification of novel substrates.

The KDM5/JARID1 sub-family are 2-oxoglutarate and Fe(II)-dependent lysine-specific histone demethylases that are characterized by their Jumonji catalytic domains. The KDM5 family is known to remove tri-/di-methyl modifications from lysine-4 of histone H3 (i.e. H3-K4me2/3), a mark associated with active gene expression. As a result, studies to date have revolved around the influence of KDM5 on disease through their ability to regulate H3-K4me2/3. Recent evidence demonstrates that KDM5 may influence disease beyond H3-K4 demethylation, making it critical to further investigate KDM5-mediated demethylation of non-histone proteins. To help identify potential non-histone substrates for the KDM5 family, we developed a library of 180 permutated peptide substrates, with sequences that are systematically altered from the wild-type H3-K4me3 substrate. From this library, we characterized recombinant KDM5A/B/C/D substrate preference and developed recognition motifs for each KDM5 demethylase. The recognition motifs developed were used to predict potential substrates for KDM5A/B/C/D and profiled to generate a list of high-ranking and medium/low-ranking substrates for further in vitro validation. Through this approach, we identified 66 high-ranking substrates in which KDM5 demethylases displayed significant in vitro activity towards.

The role of inflammation and innate immune cells in young myocardial infarction patients without traditional risk factors

Abstract Background There is a group of patients that develops myocardial infarction (MI) in the absence of standard modifiable risk factors (SMuRFs) and at a young age. Identifying the mechanism underlying the development of atherosclerosis in these patients is paramount in order to develop adequate treatment strategies. Atherosclerosis is characterized as an inflammatory disease in which monocytes play a pivotal role. Circulating monocytes of atherosclerosis patients with SMuRFs have a pro-inflammatory phenotype. At present, the contribution of systemic inflammation, or innate immune cell activation in particular, in young SMuRFless MI patients is unknown. Purpose The aim of this study was to characterize the inflammatory profile of young SMuRFless MI patients, with an in-depth focus on monocyte phenotype, function and their epigenetic profiles. Methods We recruited 20 SMuRFless patients who suffered from a recent (1–4 year ago) MI and were &amp;lt;50 years old at the time. These patients were age- and sex matched to 18 healthy controls, who did not have atherosclerosis, as determined by coronary CT-scan. Blood samples were obtained after evening out any potential immunomodulating effect of statins and aspirin by interrupting statins for two weeks in patients and starting aspirin therapy for two weeks in controls. In both groups we studied circulating inflammatory markers with proximity extension assay, circulating monocyte (subset) phenotype with flow cytometry, and cytokine production of isolated peripheral blood mononuclear cells (PBMC) after stimulation with Toll-Like Receptor-agonists. Histone marks of chromatin accessibility were assessed on the promotor regions of several inflammatory cytokines in unstimulated isolated monocytes by chromatin immunoprecipitation (ChIP). Results PBMCs from patients showed elevated cytokine production after ex vivo stimulation compared to controls (P=0.046) (Figure 1). ChIP analysis of unstimulated monocytes revealed an overall enrichment of transcriptionally permissive tri-methylation of lysine 4 on histone H3 (H3K4me3) on the promoter regions of cytokines in patients compared to controls (Figure 2). Levels of H3K4me3 on IL-10 were significantly higher in patients (p=0.04). For TNF-α and IL-6 H3K4me3 levels did not reach statistical significance (p=0.07 and p=0.13 respectively). Despite the functional hyperresponsiveness, no differences were seen in monocyte phenotype on flow cytometry analysis (not shown). Additionally there were no differences in circulating high-sensitive C-reactive protein or other circulating inflammatory markers (not shown). Conclusions Circulating monocytes in SMuRFless MI patients are characterized by pro-inflammatory behavior, which is mediated by transcriptionally permissive epigenetic modifications at the promoters of key inflammatory cytokine genes. These findings are present in the absence of active systemic inflammation. Funding Acknowledgement Type of funding sources: None.

Retinoic acid receptor gamma is required for proliferation of pancreatic cancer cells.

Despite the expectation that retinoic acid receptor could be the potential therapeutic targets for pancreatic cancers, there has been the lack of information about the role and the impact of retinoic acid receptor gamma (RARγ, RARG) on pancreatic cancer, unlike other two RARs. Herein, we applied TCGA and GEO database to show that the expression and prognosis of RARG is closely related to pancreatic cancer, which demonstrates that RARG is commonly overexpressed in human pancreatic cancer and is an independent diagnostic marker predicting the poor prognosis of pancreatic cancer patients. In addition, we demonstrated that the reduction in the expression of RARG in human pancreatic cancer cells dramatically suppress their proliferation and tumor growth in vivo, partially attributable to the downregulation of tumor-supporting biological processes such as cell proliferation, antiapoptosis and metabolism and the decreased expression of various oncogenes like MYC and STAT3. Mechanistically, RARG binds on the promoters of MYC, STAT3, and SLC2A1 which is distinguished from well-known conventionalRetinotic acid response elements (RAREs) and that the binding is likely to be responsible for the epigenetic activation in the level of chromatin, assessed by the measurement of deposition of the gene activation marker histone H3 K27 acetylation (H3K27ac) using ChIP-qPCR. In this study, we reveal that RARG plays important role in the tumorigenesis of pancreatic cancer and represents new therapeutic targets for human pancreatic cancer.

Goldilocks meets Polycomb.

The Polycomb system modulates chromatin structure to maintain gene repression during cell differentiation. Polycomb repression involves methylation of histone H3K27 (H3K27me3) by Polycomb repressive complex 2 (PRC2), monoubiquitylation of H2A (H2Aub1) by noncanonical PRC1 (ncPRC1), and chromatin compaction by canonical PRC1 (cPRC1), which is independent of its enzymatic activity. Puzzlingly, Polycomb repression also requires deubiquitylation of H2Aub1 by Polycomb repressive deubiquitinase (PR-DUB). In this issue of Genes & Development, Bonnet and colleagues (pp. 1046-1061) resolve this paradox by showing that high levels of H2Aub1 in Drosophila lacking PR-DUB activity promotes open chromatin and gene expression in spite of normal H3K27me3 levels and PRC binding. Pertinently, gene repression is restored by concomitant loss of PRC1 E3 ubiquitin ligase activity but depends on its chromatin compaction activity. These findings suggest that PR-DUB ensures just-right levels of H2Aub1 to allow chromatin compaction by cPRC1.

Mechanism of Puerariae Lobatae Radix against lung cancer by inhibiting histone demethylase LSD1

Histone lysine-specific demethylase 1(LSD1) has become a promising molecular target for lung cancer therapy. Upon the screening platform for LSD1 activity, some Chinese herbal extracts were screened for LSD1 activity inhibition, and the underlying mechanism was preliminarily investigated at both molecular and cellular levels. The results of LSD1 inhibition showed that Puerariae Lobatae Radix extract can effectively reduce LSD1 expression to elevate the expression of H3 K4 me2 and H3 K9 me2 substrates in H1975 and H1299 cells. Furthermore, Puerariae Lobatae Radix was evaluated for its anti-lung cancer activity. It had a potent inhibitory ability against the proliferation and colony formation of both H1975 and H1299 cells. Flow cytometry and DAPI staining assays indicated that Puerariae Lobatae Radix can induce the apoptosis of lung cancer cells. In addition, it can significantly suppress the migration and reverse the epithelial-mesenchymal transition(EMT) process of lung cancer cells by activating E-cadherin and suppressing the expression of N-cadherin, slug and vimentin. To sum up, Puerariae Lobatae Radix displayed a robust inhibitory activity against lung cancer, and the mechanism may be related to the down-regulation of LSD1 expression to induce the cell apoptosis and suppress the cell migration and EMT process. These findings will provide new insights into the action of Puerariae Lobatae Radix as an anti-lung cancer agent and offer new ideas for the study on the anti-cancer action of Chinese medicine based on the epigenetic modification.

Dual targeting of EZH1 and EZH2 for the treatment of malignant rhabdoid tumors

Malignant rhabdoid tumors (MRTs) are rare and highly aggressive pediatric cancers with no standard of care. MRTs are characterized by loss of SMARCB1, which results in upregulated expression of enhancer of zeste homolog 2 (EZH2), which is responsible for the methylation of lysine 27 of histone H3 (H3K27me3), leading to the repression of gene expression. Although previous reports suggest EZH2 as an effective therapeutic target, the functions of EZH1, the other homolog of EZH, in MRT remain unknown. Here, we show that EZH1, as well as EZH2, contributes to MRT cell growth and H3K27 methylation. Depletion or selective inhibition of EZH2 led to a compensatory increase in EZH1 expression, and depletion of EZH1 enhanced the effect of EZH2 inhibition. EZH1/2 dual inhibitors suppressed MRT cell growth markedly, reflecting the reduction of H3K27me3 accumulation at one of the EZH1/2 targets, the CDKN2A locus. Dual inhibition of EZH1/2 in vivo suppressed tumor growth completely, with no significant adverse effects. These findings indicate that both EZH1 and EZH2 are potential targets for MRT therapy, and that EZH1/2 dual inhibitors may be promising therapeutic strategies for MRT.

Posterior fossa ependymoma H3 K27-mutant: an integrated radiological and histomolecular tumor analysis

Posterior fossa group A ependymomas (EPN_PFA) are characterized by a loss of H3 K27 trimethylation due to either EZHIP overexpression or H3 p.K27M mutation, similar to H3 K27-altered diffuse midline gliomas (DMG), but in reverse proportions. Very little data is available in the literature concerning H3 K27M-mutant EPN_PFA. Here, we retrospectively studied a series of nine pediatric tumors initially diagnosed as H3 K27M-mutant EPN_PFA to compare them to EZHIP-overexpressing EPN_PFA in terms of radiology, follow-up, histopathology, and molecular biology (including DNA-methylation profiling). Seven tumors clustered within EPN_PFA by DNA-methylation analysis and t-distributed stochastic neighbor embedding. Among the two remaining cases, one was reclassified as a DMG and the last was unclassified. H3 K27M-mutant EPN_PFA cases were significantly older than their counterparts with an EZHIP overexpression. Radiological and histopathological central review of our seven H3 K27M-mutant EPN_PFA cases found them to be similar to their counterparts with an EZHIP overexpression. Sequencing analyses revealed HIST1H3B (n = 2), HIST1H3C (n = 2), H3F3A (n = 1), and HIST1H3D (n = 1) K27M mutations (no sequencing analysis available for the last case which was immunopositive for H3K27M). Consequently, HIST1H3C/D mutations are more frequently observed in EPN_PFA than in classic pontine DMG, H3K27-mutant. Overall survival and event-free survival of EZHIP-overexpressing and H3 K27M-mutant EPN_PFA were similar. After surgery and radiation therapy, 5/7 patients were alive at the end of the follow-up. In summary, the diagnosis of EPN_PFA must include tumor location, growth pattern, Olig2 expression, and DNA-methylation profiling before it can be differentiated from DMG, H3 K27-altered.

KDM3A promotes oral squamous cell carcinoma cell proliferation and invasion via H3K9me2 demethylation-activated DCLK1.

Oral squamous cell carcinoma (OSCC) is a frequently-diagnosed malignancy with high potential for proliferation and invasion. Histone methylation is known as a crucial mechanism that regulates pathological processes in various cancers, including OSCC. This study sought to delve into the molecular mechanism of lysine demethylase 3A (KDM3A) in OSCC cell proliferation and invasion. Expression levels of KDM3A, lysin-9 of di-methylated histone H3 (H3K9me2), and doublecortin-like kinase 1 (DCLK1) in cells were determined by reverse-transcription quantitative polymerase chain reaction or Western blot analysis. Cell proliferation and invasion were evaluated by cell counting kit-8, colony formation, and Transwell assays. The enrichment of KDM3A and H3K9me2 on the DCLK1 promoter was determined by chromatin immunoprecipitation assay. The functional rescue experiment was performed with DCLK1 overexpression vector and si-KDM3A in CAL-27 and SCC-9 cells. KDM3A was elevated in OSCC cells. KDM3A knockdown suppressed OSCC proliferation and invasion, along with increased H3K9me2 level in OSCC cells. KDM3A and H3K9me2 were enriched on the DCLK1 promoter and inhibiting H3K9me2 improved DCLK1 expression levels. DCLK1 overexpression neutralized the inhibition of KDM3A knockdown on OSCC proliferation and invasion. KDM3A facilitated OSCC proliferation and invasion by eliminating H3K9me2 to upregulate DCLK1 expression levels.

Histone methyltransferase MLL1 drives renal tubular cell apoptosis by p53-dependent repression of E-cadherin during cisplatin-induced acute kidney injury

Mixed lineage leukemia 1 (MLL1) is a histone H3 lysine 4 (H3K4) methyltransferase that interacts with WD repeat domain 5 (WDR5) to regulate cell survival, proliferation, and senescence. The role of MLL1 in the pathogenesis of acute kidney injury (AKI) is unknown. In this study, we demonstrate that MLL1, WDR5, and trimethylated H3K4 (H3K4me3) were upregulated in renal tubular cells of cisplatin-induced AKI in mice, along with increased phosphorylation of p53 and decreased expression of E-cadherin. Administration of MM102, a selective MLL1/WDR5 complex inhibitor, improved renal function and attenuated tubular injury and apoptosis, while repressing MLL1, WDR5, and H3K4me3, dephosphorylating p53 and preserving E-cadherin. In cultured mouse renal proximal tubular cells (RPTCs) exposed to cisplatin, treatment with MM102 or transfection with siRNAs for either MLL1 or WDR5 also inhibited apoptosis and p53 phosphorylation while preserving E-cadherin expression; p53 inhibition with Pifithrin-α lowered cisplatin-induced apoptosis without affecting expression of MLL1, WDR5, and H3K4me3. Interestingly, silencing of E-cadherin offset MM102’s cytoprotective effects, but had no effect on p53 phosphorylation. These findings suggest that MLL1/WDR5 activates p53, which, in turn, represses E-cadherin, leading to apoptosis during cisplatin-induced AKI. Further studies showed that MM102 effectively inhibited cisplatin-triggered DNA damage response (DDR), as indicated by dephosphorylation of ataxia telangiectasia mutated (ATM) and ATM and Rad-3 related (ATR) proteins, dephosphorylation of checkpoint kinase 1 and 2 (Chk1 and Chk2); depression of γ-H2AX; and restrained cell cycle arrest, as evidenced by decreased expression of p21 and phospho-histone H3 at serine 10 in vitro and in vivo. Overall, we identify MLL1 as a novel DDR regulator that drives cisplatin-induced RPTC apoptosis and AKI by modulating the MLL1/WDR5-/ATR/ATM-Chk-p53-E-cadherin axis. Targeting the MLL1/WDR5 complex may have a therapeutic potential for the treatment of AKI.

Dynamical modeling of the H3K27 epigenetic landscape in mouse embryonic stem cells.

The Polycomb system via the methylation of the lysine 27 of histone H3 (H3K27) plays central roles in the silencing of many lineage-specific genes during development. Recent experimental evidence suggested that the recruitment of histone modifying enzymes like the Polycomb repressive complex 2 (PRC2) at specific sites and their spreading capacities from these sites are key to the establishment and maintenance of a proper epigenomic landscape around Polycomb-target genes. Here, to test whether such mechanisms, as a minimal set of qualitative rules, are quantitatively compatible with data, we developed a mathematical model that can predict the locus-specific distributions of H3K27 modifications based on previous biochemical knowledge. Within the biological context of mouse embryonic stem cells, our model showed quantitative agreement with experimental profiles of H3K27 acetylation and methylation around Polycomb-target genes in wild-type and mutants. In particular, we demonstrated the key role of the reader-writer module of PRC2 and of the competition between the binding of activating and repressing enzymes in shaping the H3K27 landscape around transcriptional start sites. The predicted dynamics of establishment and maintenance of the repressive trimethylated H3K27 state suggest a slow accumulation, in perfect agreement with experiments. Our approach represents a first step towards a quantitative description of PcG regulation in various cellular contexts and provides a generic framework to better characterize epigenetic regulation in normal or disease situations.

Mutation of histone H3 serine 28 to alanine influences H3K27me3-mediated gene silencing in Arabidopsis thaliana.

Histone modifications are essential for chromatin activity and play an important role in many biological processes. Trimethylation of histone H3K27 (H3K27me3) is a repressive modification established by Polycomb Repressive Complex 2 (PRC2). Although the presence of the histone H3 serine 28 phosphorylation (H3S28ph) modification at adjacent amino acid residues has both positive and negative effects on Polycomb silencing in mammals, little is known about the effect of H3S28ph on H3K27me3-mediated gene silencing in plants. In this study, we show that mutating H3S28A in Arabidopsis (Arabidopsis thaliana) causes a dominant-negative effect that leads to an early-flowering phenotype by promoting the expression of flowering-promoting genes independently of abnormal cell division. While H3S28ph levels decreased due to the H3S28A mutation, H3K27me3 levels at the same loci did not increase. Moreover, we observed decreased H3K27me3 levels at some known PRC2 target genes in H3.3S28A transgenic lines, rather than the expected enhanced H3K27me3-mediated silencing. In line with the reduced H3K27me3 levels, the expression of the PRC2 catalytic subunits CURLY LEAF and SWINGER decreased. Taken together, these data demonstrate that H3.3S28 is required for PRC2-dependent H3K27me3-mediated silencing in Arabidopsis, suggesting that H3S28 has a noncanonical function in H3K27me3-mediated gene silencing.