H3.3-K27M drives neural stem cell-specific gliomagenesis in a human iPSC-derived model

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Pervasive H3K27 Acetylation Leads to ERV Expression and a Therapeutic Vulnerability in H3K27M Gliomas

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STAT3 is a biologically relevant therapeutic target in H3K27M-mutant diffuse midline glioma

BackgroundH3K27M-mutant diffuse midline glioma (DMG) is a lethal brain tumor that usually occurs in children. Despite advances in our understanding of its underlying biology, efficacious therapies are severely lacking.MethodsWe screened a library of drugs either FDA-approved or in clinical trial using a library of patient-derived H3K27M-mutant DMG cell lines with cell viability as the outcome. Results were validated for clinical relevance and mechanistic importance using patient specimens from biopsy and autopsy, patient-derived cell lines, inhibition by gene knockdown and small molecule inhibitors, and patient-derived xenografts.ResultsKinase inhibitors were highly toxic to H3K27M-mutant DMG cells. Within this class, STAT3 inhibitors demonstrated robust cytotoxic activity in vitro. Mechanistic analyses revealed one form of activated STAT3, phospho-tyrosine- 705 STAT3 (pSTAT3), was selectively upregulated in H3K27M-mutant cell lines and clinical specimens. STAT3 inhibition by CRISPR/Cas9 knockout, shRNA or small molecule inhibition reduced cell viability in vitro, and partially restored expression of the polycomb repressive mark H3K27me3, which is classically lost in H3K27M-mutant DMG. Putative STAT3-regulated genes were enriched in an H3K27M-knockout DMG cell line, indicating relative gain of STAT3 signaling in K27M-mutant cells. Treatment of patient-derived intracranial xenografts with WP1066, a STAT3 pathway inhibitor currently in clinical use for pediatric brain tumors, resulted in stasis of tumor growth, and increased overall survival. Finally, pSTAT3(Y705) was detected in circulating plasma extracellular vesicles of patients with H3K27M-mutant DMG.ConclusionsSTAT3 is a biologically relevant therapeutic target in H3K27M-mutant DMG. STAT3 inhibition should be considered in future clinical trials.     

H3K27M和Ki-67在小儿脑胶质瘤中的表达及相关性

目的:探讨H3K27M和Ki-67在小儿脑胶质瘤组织中的表达及意义。方法:收集2013年11月至2018年6月清华大学玉泉医院38例小儿脑胶质瘤和10例正常脑组织,采用免疫组织化学检测H3K27M和Ki-67表达并结合相关统计学方法进行分析。结果:H3K27M阳性表达与性别、年龄、肿瘤部位无显著性差异(P>0.05),但与肿瘤病理级别有显著性相关(P<0.05)。Ki-67在胶质瘤中阳性表达率为100%,Ki-67高标记指数与性别、年龄无显著性相关(P>0.05),与病理级别、肿瘤部位有显著性相关(P<0.05)。患儿的性别、年龄与生存时间之间无显著性差异(P>0.05),病理分级、肿瘤部位、H3K27M蛋白阳性、Ki-67高标记指数在患儿生存时间之间有显著性差异(P<0.05)。结论:小儿脑胶质瘤中H3K27M、Ki-67的表达水平对小儿脑胶质瘤的病理分级、预后具有提示意义,且H3K27M、Ki-67蛋白在胶质瘤组织中的表达具有相关性,为研究胶质瘤的发生发展及靶向治疗提供了线索。     

骨肉瘤细胞和组织H3K27me3表达与临床病理特征相关性研究

目的 组蛋白H3K27三甲基化(H3K27me3)在肿瘤发生发展过程中起着重要作用,研究发现H3K27me3在肝癌和前列腺癌等恶性肿瘤中的表达和临床病理特征存在相关性,但其在骨肉瘤中的研究相对较少.本研究分析H3K27me3在骨肉瘤细胞和组织中的表达及其与临床病理的关系,并探讨H3K27me3在骨肉瘤发生和发展中的作用和意义.方法 所有切片均来自于河北医科大学第四医院骨科2005-01-01-2011 01-01手术切除的标本.采用蛋白印迹法检测骨肉瘤MG-63、U2-OS、Sa-OS细胞系及hFOB1.19成骨细胞中H3K27me3表达的水平差异;采用免疫组织化学染色方法检测53例骨肉瘤组织及16例骨软骨瘤组织中H3K27me3的表达水平.KaplawMeier分析比较H3K27me3高表达和低表达患者生存率之间的差异.并应用Cox回归模型分析其表达水平对预后影响.结果 hFOB1.19、MG-63、U2-OS和Sa-OS中H3K27me3蛋白表达水平分别为0.37±0.06、0.86±0.06、0.79±0.07和0.83±0.05,与hFOB1.19细胞中H3K27me3表达相比,3组骨肉瘤细胞系中H3K27me3蛋白表达水平均显著升高,P=0.023;与骨软骨瘤组织相比(56.3％),H3K27me3在骨肉瘤组织中高表达(84.9％),二者差异有统计学意义(P＜0.001),并与肿瘤大小、肺转移、Enneking分期和生存率有关,P值分别为0.037、0.020、0.023和0.046.且其表达水平与患者生存期显著相关,P=0.012.结论 H3K27me3在骨肉瘤细胞及骨肉瘤组织中呈高表达,并与肿瘤的临床病理特征相关,可能会是骨肉瘤患者重要的预后因子及治疗靶点.     

H3K27M及其甲基化在弥漫性脊髓胶质瘤中的表达及与预后的关系

目的 探讨可催化组蛋白H3第27位(histone H3 lysine 27?to?methionine mutations,H3K27M)及其甲基化在弥漫性脊髓胶质瘤中的表达及其与预后的关系.方法 选择武汉大学中南医院2016年1月—2019年1月收治的75例弥漫性脊髓胶质瘤患者为研究对象,采用免疫组化和染色质免疫沉...     

H3K27me3 loss indicates an increased risk of recurrence in the Tübingen meningioma cohort

BackgroundA loss of the trimethylation of lysine 27 of histone H3 (H3K27me3) in meningioma has been recently suggested as an adjunct to identify subsets of higher risk of recurrence. The aim of the present study was to assess the prognostic value of H3K27 histone trimethylation and its potential clinical utility in the “Tübingen meningioma cohort.”MethodsPatients who underwent meningioma resection between October 2003 and December 2015 at the University Hospital Tübingen were included. Immunohistochemical stainings for H3K27me3 and the proliferation marker MIB1 were assessed and correlated with clinical parameters using univariate and multivariate Cox regressions as well as Pearson''s chi-squared and log-rank test.ResultsOverall, 1268 meningiomas were analyzed with a female to male ratio of 2.6 and a mean age of 58.7 years (range 8.3–91.0). With 163 cases lost to follow up, 1103 cases were available for further analysis with a mean follow-up of 40.3 months (range 1.1–186.3). Male gender, younger age, intracranial tumor localization, progressive tumor, subtotal resection, higher WHO grade, increased MIB1 rate, and loss of H3K27me3 were significant negative prognostic factors in the univariate analysis. H3K27me3 status and all other prognostic factors, except age and tumor location, remained significant in the multivariate model. Furthermore, adjuvant radiotherapy was an independent positive prognostic factor.ConclusionsLoss of H3K27me3 combined with MIB1 labeling index are independent prognostic factors in meningioma. These data from the Tübingen meningioma cohort support the clinical utility of H3K27me3 immunohistochemical staining in meningioma and its integration into the routine histopathological workup.     

A subset of pediatric-type thalamic gliomas share a distinct DNA methylation profile,H3K27me3 loss and frequent alteration of EGFR

BackgroundMalignant astrocytic gliomas in children show a remarkable biological and clinical diversity. Small in-frame insertions or missense mutations in the epidermal growth factor receptor gene (EGFR) have recently been identified in a distinct subset of pediatric-type bithalamic gliomas with a unique DNA methylation pattern.MethodsHere, we investigated an epigenetically homogeneous cohort of malignant gliomas (n = 58) distinct from other subtypes and enriched for pediatric cases and thalamic location, in comparison with this recently identified subtype of pediatric bithalamic gliomas.Results EGFR gene amplification was detected in 16/58 (27%) tumors, and missense mutations or small in-frame insertions in EGFR were found in 20/30 tumors with available sequencing data (67%; 5 of them co-occurring with EGFR amplification). Additionally, 8 of the 30 tumors (27%) harbored an H3.1 or H3.3 K27M mutation (6 of them with a concomitant EGFR alteration). All tumors tested showed loss of H3K27me3 staining, with evidence of overexpression of the EZH inhibitory protein (EZHIP) in the H3 wildtype cases. Although some tumors indeed showed a bithalamic growth pattern, a significant proportion of tumors occurred in the unilateral thalamus or in other (predominantly midline) locations.ConclusionsOur findings present a distinct molecular class of pediatric-type malignant gliomas largely overlapping with the recently reported bithalamic gliomas characterized by EGFR alteration, but additionally showing a broader spectrum of EGFR alterations and tumor localization. Global H3K27me3 loss in this group appears to be mediated by either H3 K27 mutation or EZHIP overexpression. EGFR inhibition may represent a potential therapeutic strategy in these highly aggressive gliomas.     

Global analysis of H3K4me3 and H3K27me3 profiles in glioblastoma stem cells and identification of SLC17A7 as a bivalent tumor suppressor gene

Epigenetic changes, including H3K4me3 and H3K27me3 histone modification, play an important role in carcinogenesis. However, no genome-wide histone modification map has been generated for gliomas. Here, we report a genome-wide map of H3K4me3 and H3K27me3 histone modifications for 8 glioma stem cell (GSC) lines, together with the associated gene activation or repression patterns. In addition, we compared the genome-wide histone modification maps of GSC lines to those of astrocytes to identify unique gene activation or repression profiles in GSCs and astrocytes. We also identified a set of bivalent genes, which are genes that are associated with both H3K4me3 and H3K27me3 marks and are poised for action in embryonic stem cells. These bivalent genes are potential targets for inducing differentiation in glioblastoma (GBM) as a therapeutic approach. Finally, we identified SLC17A7 as a bivalent tumor suppressor gene in GBM, as it is down-regulated at both the protein and RNA levels in GBM tissues compared with normal brain tissues, and it inhibits GBM cell proliferation, migration and invasion.     

Serial H3K27M cell-free tumor DNA (cf-tDNA) tracking predicts ONC201 treatment response and progression in diffuse midline glioma

BackgroundDiffuse Midline Glioma (DMG) with the H3K27M mutation is a lethal childhood brain cancer, with patients rarely surviving 2 years from diagnosis.MethodsWe conducted a multi-site Phase 1 trial of the imipridone ONC201 for children with H3K27M-mutant glioma ({"type":"clinical-trial","attrs":{"text":"NCT03416530","term_id":"NCT03416530"}}NCT03416530). Patients enrolled on Arm D of the trial (n = 24) underwent serial lumbar puncture for cell-free tumor DNA (cf-tDNA) analysis and patients on all arms at the University of Michigan underwent serial plasma collection. We performed digital droplet polymerase chain reaction (ddPCR) analysis of cf-tDNA samples and compared variant allele fraction (VAF) to radiographic change (maximal 2D tumor area on MRI).ResultsChange in H3.3K27M VAF over time (“VAF delta”) correlated with prolonged PFS in both CSF and plasma samples. Nonrecurrent patients that had a decrease in CSF VAF displayed a longer progression free survival (P = .0042). Decrease in plasma VAF displayed a similar trend (P = .085). VAF “spikes” (increase of at least 25%) preceded tumor progression in 8/16 cases (50%) in plasma and 5/11 cases (45.4%) in CSF. In individual cases, early reduction in H3K27M VAF predicted long-term clinical response (>1 year) to ONC201, and did not increase in cases of later-defined pseudo-progression.ConclusionOur work demonstrates the feasibility and potential utility of serial cf-tDNA in both plasma and CSF of DMG patients to supplement radiographic monitoring. Patterns of change in H3K27M VAF over time demonstrate clinical utility in terms of predicting progression and sustained response and possible differentiation of pseudo-progression and pseudo-response.     

EZH2和H3K27me3在食管鳞状细胞癌中的表达及意义

目的 分析EZH2和H3K27me3在食管鳞状细胞癌（esophageal squamous cell carcinoma, ESCC）组织中的表达及与食管鳞状细胞癌临床病理指标之间的关系,并评价其表达对食管鳞状细胞癌患者预后的意义。方法 利用免疫组织化学法检测65例ESCC患者癌组织及相应的癌旁组织中EZH2及H3K27me3的蛋白表达,分析两者表达与ESCC患者临床病理学指标之间的相关性,并分析两者之间表达的相关性。利用Kaplan-Meier方法及Log rank检验分析两者表达与食管癌生存率之间的关系。结果 癌旁组织中EZH2和H3K27me3蛋白阳性表达率分别为44.6%和26.2%,ESCC组织中分别为70.8%和47.7%,EZH2和H3K27me3蛋白表达均与ESCC患者的组织学分级及淋巴结转移状态呈正相关关系,且EZH2和H3K27me3蛋白表达之间存在正相关关系。EZH2和H3K27me3表达阳性的ESCC患者五年总生存率均明显低于EZH2和H3K27me3表达阴性的患者;EZH2和H3K27me3双阳性的ESCC患者五年总生存率明显低于EZH2和H3K27me3单阳性和双阴性的ESCC患者。Cox回归结果显示,淋巴结转移、组织学分级、EZH2和H3K27me3表达是ESCC患者的独立预后因素。结论 EZH2和H3K27me3在ESCC中均高表达,并与ESCC患者的组织学分级和淋巴结转移状态相关,可能是ESCC患者的不良预后因子。     

Global H3K27 trimethylation and EZH2 abundance in breast tumor subtypes

Polycomb repressive complex 2 (PRC2) and its core member enhancer of zeste homolog 2 (EZH2) mediate the epigenetic gene silencing mark: trimethylation of lysine 27 on histone 3 (H3K27me3). H3K27me3 is characteristic of the chromatin at genes involved in developmental regulation in undifferentiated cells. Overexpression of EZH2 has been found in several cancer types such as breast, prostate, melanoma and bladder cancer. Moreover, overexpression is associated with highly proliferative and aggressive types of breast and prostate tumors. We have analyzed the abundance of EZH2 and H3K27me3 using immunohistochemistry in two large and well-characterized breast tumor data sets encompassing more than 400 tumors. The results have been analyzed in relation to the molecular subtypes of breast tumors (basal-like, luminal A, luminal B, HER2-enriched and normal-like), as well as in subtypes defined by clinical markers (triple negative, ER+/HER2-/Ki67low, ER+/HER2-/Ki67high and HER2+), and were validated in representative breast cancer cell lines by western blot. We found significantly different expression of both EZH2 and H3K27me3 across all subtypes with high abundance of EZH2 in basal-like, triple negative and HER2-enriched tumors, and high H3K27me3 in luminal A, HER2-enriched and normal-like tumors. Intriguingly, the two markers show an inverse correlation, particularly for the basal-like and triple negative tumors. Consequently, high expression of EZH2 was associated with poor distant disease-free survival whereas high expression of H3K27me3 was associated with better survival. Additionally, none of 182 breast tumors was found to carry a previously described EZH2 mutation affecting Tyr641. Our observation that increased expression of EZH2 does not necessarily correlate with increased abundance of H3K27me3 supports the idea that EZH2 can have effects beyond epigenetic silencing of target genes in breast cancer.     

The Value of H3K27me3 Immunohistochemistry in Differentiating Malignant Peripheral Nerve Sheath Tumour with Its Histologic Mimickers

Background: Diagnosis of malignant peripheral nerve sheath tumor (MPNST) is rather challenging due to its divergent morphologic heterogeneity and lack of specific ancillary test. The emergence of H3K27 trimethylation (H3K27me3) as a new immunohistochemistry (IHC) marker for MPNST have recently available to assist pathologists in differentiating MPNST from other histologic mimics. We aim to study the expression pattern of H3K27me3 in MPNST and its histologic mimickers and their association with the clinicopathological data. Methodology: A total of 59 benign and malignant spindle cell tumours (18 MPNST and 41 of its histologic mimickers which included 10 schwannoma, 13 neurofibroma, 4 synovial sarcoma, 3 fibrosarcoma, 2 gastrointestinal stromal tumour (GIST), 4 leiomyosarcoma, 1 spindle cell liposarcoma, 1 solitary fibrous tumour, 2 low grade fibromyxoid sarcoma and 1 unclassified spindle cell sarcoma), diagnosed from January 1998 to April 2018 in Hospital Universiti Sains Malaysia (HUSM) were tested for H3K27me3 by IHC. The MPNST histological grade was assessed based on the French Fe’de’ ration Nationale des Centres de LutteContre le Cancer (FNCLCC) for 3 tiers system (low grade, intermediate grade and high grade). The clinicopathological data were retrieved from the patients’ record. Results: A total of 61.1% (11/18 MPNST) showed loss of H3K27me3 expression which is statistically significant as compared to its histologic mimics (p<0.001). Similar findings (p=0.026) were also observed in high grade MPNST (81.8%), intermediate grade MPNST (100%) and 0% in low grade MPNST. Conclusion: H3K27me3, combined with other panel of markers, is useful in MPNST diagnosis to differentiate it from the histological mimickers.     

Loss of H3K27me3 in meningiomas

BackgroundThere is a critical need for objective and reliable biomarkers of outcome in meningiomas beyond WHO classification. Loss of H3K27me3 has been reported as a prognostically unfavorable alteration in meningiomas. We sought to independently evaluate the reproducibility and prognostic value of H3K27me3 loss by immunohistochemistry (IHC) in a multicenter study.MethodsIHC staining for H3K27me3 and analyses of whole slides from 181 meningiomas across three centers was performed. Staining was analyzed by dichotomization into loss and retained immunoreactivity, and using a 3-tiered scoring system in 151 cases with clear staining. Associations of grouping with outcome were performed using Kaplan-Meier survival estimates.ResultsA total of 21 of 151 tumors (13.9%) demonstrated complete loss of H3K27me3 staining in tumor with retained endothelial staining. Overall, loss of H3K27me3 portended a worse outcome with shorter times to recurrence in our cohort, particularly for WHO grade 2 tumors which were enriched in our study. There were no differences in recurrence-free survival (RFS) for WHO grade 3 patients with retained vs loss of H3K27me3. Scoring by a 3-tiered system did not add further insights into the prognostic value of this H3K27me3 loss. Overall, loss of H3K27me3 was not independently associated with RFS after controlling for WHO grade, extent of resection, sex, age, and recurrence status of tumor on multivariable Cox regression analysis.ConclusionsLoss of H3K27me3 identifies a subset of WHO grade 2 and possibly WHO grade 1 meningiomas with increased recurrence risk. Pooled analyses of a larger cohort of samples with standardized reporting of clinical definitions and staining patterns are warranted.     

H3K27甲基化与EZH2在B细胞非霍奇金淋巴瘤中表观遗传调控的研究进展

异常的组蛋白修饰与DNA甲基化是表观遗传学研究的重要领域,两者相互作用可导致特定基因的转录激活或抑制。H3K27的甲基化水平及其甲基转移酶EZH2可能与B细胞非霍奇金淋巴瘤（B-cell NHL）的特殊分型及不良预后密切相关。本文着重介绍H3K27me3与EZH2在B细胞非霍奇金淋巴瘤中的发生和发展过程的作用及相应机制。     

EZH2和H3K27me3的表达对食管癌细胞迁移和侵袭能力的影响

目的:通过转染Zeste同源物增强子2(enhancer of zeste homolog 2,EZH2)过表达或者敲低载体,探讨EZH2和Lys27位点三甲基化组蛋白H3(histone H3 methylated Lys27,H3K27me3)对食管麟状细胞癌(esophageal squamous cell cancer,ESCC)细胞迁移和侵袭能力的影响.方法:应用实时荧光定量PCR、Western blotting法检测ESCC细胞株KYSE30、KYSE170、TE1、Eca109中EZH2 mRNA水平,以及ESCC细胞过表达或者敲低EZH2对H3 K27me3表达水平的影响.用划痕实验及Transwell侵袭实验分析过表达或者敲低EZH2后ESCC细胞的迁移侵袭能力.用实时荧光定量PCR法分析ESCC细胞过表达及敲低EZH2对MMPs mRNA水平的影响.结果:食管癌Eea109及TE1细胞中EZH2和H3K27me3 mRNA和蛋白水平明显高于KYSE30及KYSE170细胞(P＜0.05).过表达EZH2的食管癌KYSE30及KYSE170细胞H3K27me3蛋白的表达水平显著升高(P＜0.05),敲低EZH2后Eca109及TE1细胞H3 K27 me3蛋白的表达水平明显降低(P＜0.05).过表达EZH2后,KYSE30及KYSE170细胞的穿膜数目明显增多[(281.33±4.10)、(241.67 ±4.04) vs(132.00 ±4.00)、(105.33 ±3.51)个,均P＜0.05]、迁移距离明显增大[(63.6±1.2)、(62.5±2.5)vs (23.0±2.3)、(21.2±1.0) μm,P＜0.05].敲低EZH2后Eca109及TE1细胞的穿膜数目显著减少(均P＜0.05),转染shEZH2后Eca109及TE1细胞迁移的距离明显减小(均P＜0.05).结论:EZH2可增加靶基因启动子上组蛋白H3第27位赖氨酸的三甲基化,并增强ESCC细胞的迁移和侵袭能力.     

Global histone modification of H3K27 correlates with the outcomes in patients with metachronous liver metastasis of colorectal cancer

Background

We evaluated the methylation patterns of histone H3 lysine 27 (H3K27), H3 lysine 36 (H3K36) and the expression of H3K27 methylase EZH2 in patients with colorectal carcinomas with metachronous liver metastasis to search for biomarkers identifying these patients.

Methods

Double 2-mm core tissue microarrays were made from 54 paraffin-embedded samples of primary colorectal adenocarcinomas and corresponding liver metastases and examined using an immunohistochemical analysis of dimethylation and trimethylation in H3K27, H3K36 and EZH2. Positive tumor cell staining for each histone modification (H-score) was used to classify patients into low- and high-staining groups, which were then examined to identify any correlations between the clinicopathological parameters and the clinical outcomes.

Results

The H-scores of H3K27me2 were lower in the liver metastases than in the corresponding primary tumors, while the H-scores of H3K36me2 were higher in the liver metastases than in the corresponding primary tumors (P < 0.001). H3K27me2 in the primary tumors correlated with tumor size (P = 0.016), H3K36me2 in the primary tumors correlated with histological type (P = 0.038), and H3K36me3 in the primary tumors correlated with lymph node metastasis (P = 0.017). In addition, lower levels of H3K27me2 in the primary tumors correlated with poorer survival rates (P = 0.039). The multivariate survival analysis showed that the H3K27me2 status is an independent prognostic factor for colorectal cancer patients (P = 0.047).

Conclusions

Our findings suggest that the methylation level of H3K27me2 detected with immunohistochemistry may be an independent prognostic factor for metachronous liver metastasis of colorectal carcinomas.     

胶质瘤中PI3K、AKT2和NDRG2mRNA的表达及相关性

目的：研究胶质瘤中蛋白激酶P13K、AKT2 mRNA表达与NDRG2 mRNA表达的相关性，寻找与NDRG2相互作用的上下游分子。方法：用实时定量PCR技术检测29例脑胶质瘤及相应瘤旁组织中P13K、AKT2和NDRG2 mRNA表达量。结果：29例脑胶质瘤NDRG2 mRNA表达明显低于瘤旁组织（P〈0．05），且不同病理分级间表达有显著性差异（P〈0．05），其中Ⅳ级NDRG2 mRNA表达量明显低于Ⅱ级和Ⅲ级胶质瘤（P=0．007，P=0．03）；AKT2，P13K mRNA表达在胶质瘤及瘤旁组织之间无明显差异（P〉0．05）。AKT2，P13K mRNA表达与NDRG2 mRNA表达的相关性无统计学意义（P〉0．05）。结论：NDRG2可能与胶质瘤的发生发展及恶性演变有关，在mRNA表达水平，还不能认为P13K、AKT2与NDRG2之间有相关性。