SETD1A function in leukemia is mediated through interaction with mitotic regulators BuGZ/BUB3.

The H3K4 methyltransferase SETD1A plays a crucial role in leukemia cell survival through its noncatalytic FLOS domain-mediated recruitment of cyclin K and regulation of DNA damage response genes. In this study, we identify a functional nuclear localization signal in and interaction partners of the FLOS domain. Our screen for FLOS domain-binding partners reveals that the SETD1A FLOS domain binds mitosis-associated proteins BuGZ/BUB3. Inhibition of both cyclin K and BuGZ/BUB3-binding motifs in SETD1A shows synergistic antileukemic effects. BuGZ/BUB3 localize to SETD1A-bound promoter-TSS regions and SETD1A-negative H3K4me1-positive enhancer regions adjacent to SETD1A target genes. The GLEBS motif and intrinsically disordered region of BuGZ are required for both SETD1A-binding and leukemia cell proliferation. Cell-cycle-specific SETD1A restoration assays indicate that SETD1A expression at the G1/S phase of the cell cycle promotes both the expression of DNA damage response genes and cell cycle progression in leukemia cells.

Tumorigenic activation around HPV integrated sites in head and neck squamous cell carcinoma.

Human papillomavirus (HPV) is causally involved in the development of head and neck squamous cell carcinoma (HNSCC). The integration of HPV drives tumorigenesis through expression of oncogenic viral genes as well as genomic alterations in surrounding regions. To elucidate involvement of epigenetic dysregulation in tumorigenesis, we here performed integrated analyses of the epigenome, transcriptome and interactome using ChIP-seq, RNA-seq and Hi-C and 4C-seq for HPV(+) HNSCCs. We analyzed clinical HNSCC using The Cancer Genome Atlas data and found that genes neighboring HPV integration sites were significantly upregulated and were correlated with oncogenic phenotypes in HPV(+) HNSCCs. While we found four HPV integration sites in HPV(+) HNSCC cell line UPCI-SCC-090 through target enrichment sequencing, 4C-seq revealed 0.5 to 40 Mb of HPV-interacting regions (HPVIRs) where host genomic regions interacted with integrated HPV genomes. While 9% of the HPVIRs were amplified and activated epigenetically forming super-enhancers, the remaining non-amplified regions were found to show a significant increase in H3K27ac levels and an upregulation of genes associated with GO terms, for example, Signaling by WNT and Cell Cycle. Among those genes, ITPR3 was significantly upregulated, involving UPCI-SCC-090-specific super-enhancer formation around the ITPR3 promoter and in the 80-kb-downstream region. The knockdown of ITPR3 by siRNA or CRISPR deletions of the distant enhancer region led to a significant suppression of cell proliferation. The epigenetic activation of HPVIRs was also confirmed in other cell lines, UM-SCC-47 and UM-SCC-104. These data indicate that epigenetic activation in HPVIRs contributes, at least partially, to genesis of HPV(+) HNSCC.

Association of frequent hypermethylation with high grade histological subtype in lung adenocarcinoma.

Lung adenocarcinoma is classified morphologically into five histological subtypes according to the WHO classification. While each histological subtype correlates with a distinct prognosis, the molecular basis has not been fully elucidated. Here we conducted DNA methylation analysis of 30 lung adenocarcinoma cases annotated with the predominant histological subtypes and three normal lung cases using the Infinium BeadChip. Unsupervised hierarchical clustering analysis revealed three subgroups with different methylation levels: high-, intermediate-, and low-methylation epigenotypes (HME, IME, and LME). Micropapillary pattern (MPP)-predominant cases and those with MPP components were significantly enriched in HME (p = 0.02 and p = 0.03, respectively). HME cases showed a significantly poor prognosis for recurrence-free survival (p < 0.001) and overall survival (p = 0.006). We identified 365 HME marker genes specifically hypermethylated in HME cases with enrichment of "cell morphogenesis" related genes; 305 IME marker genes hypermethylated in HME and IME, but not in LME, with enrichment "embryonic organ morphogenesis"-related genes; 257 Common marker genes hypermethylated commonly in all cancer cases, with enrichment of "regionalization"-related genes. We extracted surrogate markers for each epigenotype and designed pyrosequencing primers for five HME markers (TCERG1L, CXCL12, FAM181B, HOXA11, GAD2), three IME markers (TBX18, ZNF154, NWD2) and three Common markers (SCT, GJD2, BARHL2). DNA methylation profiling using Infinium data was validated by pyrosequencing, and HME cases defined by pyrosequencing results also showed the worse recurrence-free survival. In conclusion, lung adenocarcinomas are stratified into subtypes with distinct DNA methylation levels, and the high-methylation subtype correlated with MPP-predominant cases and those with MPP components and showed a poor prognosis.

Adenocarcinoma of the stomach and esophagogastric junction with low DNA methylation show poor prognoses.

BACKGROUND: Gastric cancer (GC) is characterized by unique DNA methylation epigenotypes (MEs). However, MEs including adenocarcinomas of the esophagogastric junction (AEG) and background non-neoplastic columnar mucosae (NM) remain to be clarified. METHODS: We analyzed the genome-wide DNA MEs of AEG, GC, and background NM using the Infinium 450 k beadarray, followed by quantitative pyrosequencing validation. Large-scale data from The Cancer Genome Atlas (TCGA) were also reviewed. RESULTS: Unsupervised two-way hierarchical clustering using Infinium data of 21 AEG, 30 GC, and 11 NM revealed four DNA MEs: extremely high-ME (E-HME), high-ME (HME), low-ME (LME), and extremely low-ME (E-LME). Promoter methylation levels were validated by pyrosequencing in 146 samples. Non-inflammatory normal mucosae were clustered into E-LME, whereas gastric or esophagogastric junction mucosae with chronic inflammatory changes caused by either Helicobacter pylori infection or reflux esophagitis were clustered together into LME, suggesting that inflammation status determined DNA MEs regardless of the cause. Three cases of Barrett's-related adenocarcinoma were clustered into HME. Among 94 patients whose tumors could be clustered into one of four MEs, 11 patients with E-LME cancers showed significantly shorter overall survival than that in the other MEs, even with the multivariate Cox regression estimate. TCGA data also showed enrichment of AEG in HME and a poorer prognosis in E-LME. CONCLUSIONS: E-LME cases, newly confirmed in this study, form a unique subtype with poor prognosis that is not associated with inflammation-associated elevation of DNA methylation levels. LME could be acquired via chronic inflammation, regardless of the cause, and AEG might preferentially show HME.

The Link of mRNA and rRNA Transcription by PUF60/FIR through TFIIH/P62 as a Novel Therapeutic Target for Cancer.

The interaction between mRNA and ribosomal RNA (rRNA) transcription in cancer remains unclear. RNAP I and II possess a common N-terminal tail (NTT), RNA polymerase subunit RPB6, which interacts with P62 of transcription factor (TF) IIH, and is a common target for the link between mRNA and rRNA transcription. The mRNAs and rRNAs affected by FUBP1-interacting repressor (FIR) were assessed via RNA sequencing and qRT-PCR analysis. An FIR, a c-myc transcriptional repressor, and its splicing form FIRΔexon2 were examined to interact with P62. Protein interaction was investigated via isothermal titration calorimetry measurements. FIR was found to contain a highly conserved region homologous to RPB6 that interacts with P62. FIRΔexon2 competed with FIR for P62 binding and coactivated transcription of mRNAs and rRNAs. Low-molecular-weight chemical compounds that bind to FIR and FIRΔexon2 were screened for cancer treatment. A low-molecular-weight chemical, BK697, which interacts with FIRΔexon2, inhibited tumor cell growth with rRNA suppression. In this study, a novel coactivation pathway for cancer-related mRNA and rRNA transcription through TFIIH/P62 by FIRΔexon2 was proposed. Direct evidence in X-ray crystallography is required in further studies to show the conformational difference between FIR and FIRΔexon2 that affects the P62-RBP6 interaction.

Genetic alterations in squamous cell lung cancer associated with idiopathic pulmonary fibrosis.

Patients with idiopathic pulmonary fibrosis (IPF) are at higher risk of developing lung cancers including squamous cell lung carcinoma (SCC), which typically carries a poor prognosis. Although the molecular basis of cancer development subsequent to IPF has not been fully investigated, we recently reported two epigenetic phenotypes characterized by frequent and infrequent DNA hypermethylation in SCC, and an association of the infrequent hypermethylation phenotype with IPF-associated SCCs. Here, we conducted targeted exon sequencing in SCCs with and without IPF using the Human Lung Cancer Panel to investigate the genetic basis of IPF-associated SCC. SCCs with and without IPF displayed comparable numbers of total mutations (137 ± 22 vs 131 ± 27, P = .5), nonsynonymous mutations (72 ± 14 vs 69 ± 16, P = .5), indels (3.0 ± 3.5 vs 3.0 ± 3.9, P = 1) and synonymous mutations (62 ± 9.1 vs 60 ± 12, P = .5). Signature 1 was the predominant signature in SCCs with and without IPF. SETD2 and NFE2L2 mutations were significantly associated with IPF (44% vs 13%, P = .03 for SETD2; 38% vs 10%, P = .04 for NFE2L2). MYC amplification, assessed by copy number variant analysis, was also significantly associated with IPF (18.8% vs 0%, P = .04). Mutations in TP53 and CDKN2A were observed relatively frequently in SCCs with frequent hypermethylation (P = .02 for TP53 and P = .06 for CDKN2A). Survival analysis revealed that the SETD2 mutation was significantly associated with worse prognosis (P = .04). Collectively, we found frequent involvement of SETD2 and NFE2L2 mutations and MYC amplification in SCCs with IPF, and an association of a SETD2 mutation with poorer prognosis.

Anti-FIRΔexon2 autoantibody as a novel indicator for better overall survival in gastric cancer.

There is no clinically available biomarker for efficiently indicating the overall survival or therapy response of gastric cancer (GC). The autoantibodies (Abs) in the sera of anti-far-upstream element-binding protein-interacting repressor-lacking exon2 (FIRΔexon2), anti-sorting nexin 15, and anti-spermatogenesis and oogenesis-specific basic helix-loop-helix 1 were markedly higher in GC patients than in healthy donors (HDs). These Abs were identified by large-scale serological identification of antigens by recombinant cDNA expression cloning screenings and their expression levels were evaluated by amplified luminescence proximity homogeneous assay. In particular, compared with age-matched HDs, the level of anti-FIRΔexon2 Abs in GC patients was significantly higher (P < .001). The Spearman's rank correlation analysis between anti-FIRΔexon2 Abs and clinically available tumor markers such as carcinoembryonic antigen (CEA) was statistically insignificant, indicating that FIRΔexon2 Abs is an independent biomarker. We performed receiver-operating curve analysis to evaluate the anti-FIRΔexon2 Ab as a candidate biomarker with CEA and carbohydrate antigen 19-9 (CA19-9). The overall survival of GC patients with high anti-FIRΔexon2 Abs titer was significantly favorable (P = .04) than that of GC patients who were below detection level of anti-FIRΔexon2 Abs. However, clinical stages were not apparently correlated with the levels of anti-FIRΔexon2 Ab, CEA, and CA19-9. In conclusion, anti-FIRΔexon2 Abs detected in GC patients is a potential biomarker for monitoring a better prognosis. Hence, anti-FIRΔexon2 Abs is a promising biomarker for indicating better overall survival of gastric cancer patients.

Activation of EHF via STAT3 phosphorylation by LMP2A in Epstein-Barr virus-positive gastric cancer.

Epstein-Barr virus (EBV) is associated with approximately 10% of gastric cancers (GCs). We previously showed that EBV infection of gastric epithelial cells induces aberrant DNA methylation in promoter regions, which causes silencing of critical tumor suppressor genes. Here, we analyzed gene expressions and active histone modifications (H3K4me3, H3K4me1, and H3K27ac) genome-widely in EBV-positive GC cell lines and in vitro EBV-infected GC cell lines to elucidate the transcription factors contributing to tumorigenesis through enhancer activation. Genes associated with "signaling of WNT in cancer" were significantly enriched in EBV-positive GC, showing increased active ß-catenin staining. Genes neighboring activated enhancers were significantly upregulated, and EHF motif was significantly enriched in these active enhancers. Higher expression of EHF in clinical EBV-positive GC compared with normal tissue and EBV-negative GC was confirmed by RNA-seq using The Cancer Genome Atlas cohort, and by immunostaining using our cohort. EHF knockdown markedly inhibited cell proliferation. Moreover, there was significant enrichment of critical cancer pathway-related genes (eg, FZD5) in the downstream of EHF. EBV protein LMP2A caused upregulation of EHF via phosphorylation of STAT3. STAT3 knockdown was shown to inhibit cellular growth of EBV-positive GC cells, and the inhibition was rescued by EHF overexpression. Our data highlighted the important role of EBV infection in gastric tumorigenesis via enhancer activation.

Genetic subtype classification using a simplified algorithm and mutational characteristics of diffuse large B-cell lymphoma in a Japanese cohort.

Recent large-scale genetic studies have proposed a new genetic classification of diffuse large B-cell lymphoma (DLBCL), which is clinically and biologically heterogeneous. However, the classification methods were complicated to be introduced into clinical practice. Here we retrospectively evaluated the mutational status and copy number changes of 144 genes in 177 Japanese patients with DLBCL, using targeted DNA sequencing. We developed a simplified algorithm for classifying four genetic subtypes-MYD88, NOTCH2, BCL2, and SGK1-by assessing alterations in 18 representative genes and BCL2 and BCL6 rearrangement status, integrating the significant genes from previous studies. In our cohort and another validation cohort from published data, the classification results in our algorithm showed close agreement with the other established algorithm. A differential prognosis among the four groups was observed. The NOTCH2 group showed a particularly poorer outcome than similar groups in previous reports. Furthermore, our study revealed unreported genetic features in the DLBCL subtypes that are mainly reported in Japanese patients, such as CD5-positive DLBCL and methotrexate-associated lymphoproliferative disorders. These results indicate the utility of our simplified method for DLBCL genetic subtype classification, which can facilitate the optimisation of treatment strategies. In addition, our study highlights the genetic features of Japanese patients with DLBCL.

Stratification of HPV-associated and HPV-negative oropharyngeal squamous cell carcinomas based on DNA methylation epigenotypes.

While the incidence of oropharyngeal squamous cell carcinoma (OPSCC) has been increasing in these two decades, primarily due to human papillomavirus (HPV), stratification of OPSCC into molecular subgroups showing different clinicopathological features has not been fully investigated. We performed DNA methylome analysis using Infinium 450k for 170 OPSCC cases, including 89 cases in our cohort and 81 cases reported by The Cancer Genome Atlas, together with targeted exon sequencing analysis. We stratified OPSCC by hierarchical clustering analysis using methylome data. Methylation levels of classifier markers were validated quantitatively using pyrosequencing, and area under the curve (AUC) values of receiver operating characteristics (ROC) curves were calculated. OPSCC was stratified into four epigenotypes: HPV(+) high-methylation (OP1), HPV(+) intermediate-methylation (OP2), HPV(-) intermediate-methylation (OP3) and HPV(-) low-methylation (OP4). Ten methylation marker genes were generated: five to classify HPV(+) cases into OP1 and OP2, and five to classify HPV(-) cases into OP3 and OP4. AUC values of ROC curves were 0.969 and 0.952 for the two marker panels, respectively. While significantly higher TP53 mutation and CCND1 copy number gains were observed in HPV(-) than in HPV(+) groups (p < 0.01), no significant difference of genomic aberrations was observed between OP1 and OP2, or OP3 and OP4. The four epigenotypes showed significantly different prognosis (p = 0.0006), distinguishing the most favorable OPSCC subgroup (OP1) among generally favorable HPV(+) cases, and the most unfavorable OPSCC subgroup (OP3) among generally unfavorable HPV(-) cases. HPV(+) and HPV(-) OPSCC are further divided into distinct DNA methylation epigenotypes, showing significantly different prognosis.

A low DNA methylation epigenotype in lung squamous cell carcinoma and its association with idiopathic pulmonary fibrosis and poorer prognosis.

Patients with idiopathic pulmonary fibrosis (IPF) have higher risk of developing lung cancer, for example, squamous cell carcinoma (SCC), and show poor prognosis, while the molecular basis has not been fully investigated. Here we conducted DNA methylome analysis of lung SCC using 20 SCC samples with/without IPF, and noncancerous lung tissue samples from smokers/nonsmokers, using Infinium HumanMethylation 450K array. SCC was clustered into low- and high-methylation epigenotypes by hierarchical clustering analysis. Genes hypermethylated in SCC significantly included genes targeted by polycomb repressive complex in embryonic stem cells, and genes associated with Gene Ontology terms, for example, "transcription" and "cell adhesion," while genes hypermethylated specifically in high-methylation subgroup significantly included genes associated with "negative regulation of growth." Low-methylation subgroup significantly correlated with IPF (78%, vs. 17% in high-methylation subgroup, p = 0.04), and the correlation was validated by additional Infinium analysis of SCC samples (n = 44 in total), and data from The Cancer Genome Atlas (n = 390). The correlation between low-methylation subgroup and IPF was further validated by quantitative methylation analysis of marker genes commonly hypermethylated in SCC (HOXA2, HOXA9 and PCDHGB6), and markers specifically hypermethylated in high-methylation subgroup (DLEC1, CFTR, MT1M, CRIP3 and ALDH7A1) in 77 SCC cases using pyrosequencing (p = 0.003). Furthermore, low-methylation epigenotype significantly correlated with poorer prognosis among all SCC patients, or among patients without IPF. Multivariate analysis showed that low-methylation epigenotype is an independent predictor of poor prognosis. These may suggest that lung SCC could be stratified into molecular subtypes with distinct prognosis, and low-methylation lung SCC that significantly correlates with IPF shows unfavorable outcome.

High expression of CXCL14 is a biomarker of lung adenocarcinoma with micropapillary pattern.

Lung adenocarcinoma with micropapillary pattern (MPP) has an aggressive malignant behavior. Limited resection should be avoided because of its high recurrence rate. If adenocarcinoma with MPP is diagnosed preoperatively, the selection of proper treatment is possible. To explore a preoperative biomarker for diagnosing MPP, we undertook RNA sequencing analysis of 25 clinical samples as the training set, including 6 MPP, 16 other adenocarcinoma subtypes, and 3 normal lung tissues. Unsupervised hierarchical clustering analysis suggested a presence of subgroup with MPP showing different gene expression phenotype. We extracted differentially expressed genes with high expression levels in MPP samples, and chose VSIG1, CXCL14, and BAMBI as candidate biomarkers for MPP. Reverse transcription-quantitative PCR analysis confirmed a significantly higher expression of VSIG1 (P = .03) and CXCL14 (P = .02) in MPP than others. In a validation set of 4 MPP and 4 non-MPP samples, CXCL14 expression was validated to be significantly higher in MPP than in non-MPP (P = .04). Comparing a total of 10 MPP and 20 non-MPP samples, the area under the curve of CXCL14 to distinguish MPP from others was 0.89. The threshold value was 0.0116, corresponding to sensitivity 80% and specificity 90%. In immunostaining of CXCL14, the staining score was significantly higher in MPP cases than others, where not only the MPP component but also other components showed heterogeneous staining in adenocarcinoma tissues with MPP. Moreover, a higher staining score of CXCL14 was significantly associated with poorer prognosis in all patients (P = .01) or within cases in stage I-III (P = .01). In summary, we identified CXCL14 as a possible diagnostic biomarker of MPP.

Establishment of epigenetic markers to predict irradiation efficacy against oropharyngeal cancer.

Irradiation, or chemoradiotherapy, is a curative treatment for oropharyngeal squamous cell carcinoma (OPSCC). Its invasiveness, however, can often negate its efficacy. Therefore, developing methods to predict which patients would benefit from irradiation is urgent. Promoter DNA hypermethylation was recently reported to correlate with favorable OPSCC prognosis. It is still unclear, however, whether there is an association between promoter DNA methylation and response to irradiation. In this study, we analyzed DNA methylation in the specimens from 40 OPSCC patients who had undergone irradiation, using the Infinium assay. Our results showed significant correlation between high levels of promoter DNA methylation and better response to treatment (P < 0.01). We used the 10 most differentially-methylated genes between responders and non-responders to develop a panel of predictive markers for efficacy. Our panel had high sensitivity, specificity and accuracy (92%, 93% and 93%, respectively). We conducted pyrosequencing to quantitatively validate the methylation levels of 8 of the 10 marker genes (ROBO1, ULK4P3, MYOD1, LBX1, CACNA1A, IRX4, DPYSL3 and ELAVL2) obtained by Infinium. The validation by pyrosequencing showed that these 8 genes had a high prediction performance for the training set of 40 specimens and for a validation set of 35 OPSCC specimens, showing 96% sensitivity, 89% specificity and 94% accuracy. Methylation of these markers correlated significantly with better progression-free and overall survival rates, regardless of human papillomavirus status. These results indicate that increased DNA methylation is associated with better responses to irradiation therapy and that DNA methylation can help establish efficacy prediction markers in OPSCC.

Anti-FIRΔexon2, a splicing variant form of PUF60, autoantibody is detected in the sera of esophageal squamous cell carcinoma.

Anti-PUF60 autoantibodies are reportedly detected in the sera of patients with dermatomyositis and Sjögren's syndrome; however, little is known regarding its existence in the sera of cancer patients. FIR, a splicing variant of the PUF60 gene, is a transcriptional repressor of c-myc. In colorectal cancer, there is an overexpression of the dominant negative form of FIR, in which exon 2 is lacking (FIRΔexon2). Previously, large-scale SEREX (serological identification of antigens by recombinant cDNA expression cloning) screenings have identified anti-FIR autoantibodies in the sera of cancer patients. In the present study, we revealed the presence and significance of anti-FIR (FIR/FIRΔexon2) Abs in the sera of patients with esophageal squamous cell carcinoma (ESCC). Our results were validated by an amplified luminescence proximity homogeneous assay using sera of patients with various cancer types. We revealed that anti-FIRΔexon2 Ab had higher sensitivity than anti-FIR Ab. Receiver operating characteristic (ROC) analysis was applied for evaluating the use of anti-FIRΔexon2 Ab as candidate markers such as anti-p53 Ab and carcinoembryonic antigen, and the highest area under the ROC curve was observed in the combination of anti-FIRΔexon2 Ab and anti-p53 Ab. In summary, our results suggest the use of anti-FIRΔexon2 Ab in combination with the anti-p53 Ab as a predictive marker for ESCC. The area under the ROC curve was further increased in the advanced stage of ESCC. The value of anti-FIRΔexon2 autoantibody as novel clinical indicator against ESCC and as a companion diagnostic tool is discussed.

Clonal immunoglobulin λ light-chain gene rearrangements detected by next generation sequencing in POEMS syndrome.

Polyneuropathy, organomegaly, endocrinopathy, M-protein, and skin changes (POEMS) syndrome is a rare plasma cell dyscrasia characterized by polyneuropathy, organomegaly, endocrinopathy, extravascular fluid overload, M protein, and a myriad of skin changes. The pathogenesis is poorly understood, but monoclonal plasma cells are λ-restricted and these immunoglobulin λ light chain variable (IGLV) region genes are derived from only two germlines, either IGLV1-44 or 1-40. Here we analyzed the clonal IGLV gene rearrangements of genomic DNA samples of bone marrow mononuclear cells using next-generation sequencing (NGS) to understand the clonal composition of IGLV genes in patients with POEMS syndrome (n = 30). The dominant IGLV gene rearrangement of POEMS syndrome-specific germline sequences were significantly increased in 11 POEMS patients (36.7%; IGLV1-44: n = 9, IGLV1-40: n = 2). In some cases, IGLV gene rearrangement clone was not detected as significant increase but was detected using cDNA samples by heteroduplex (HD) analysis and Sanger sequencing, suggesting that the quite small number of monoclonal plasma cells may produce large quantity of mRNA of monoclonal proteins. However, significant increase of dominant clone sizes was not directly linked to the initial disease status. On the other hand, in cases with significantly increased dominant clones, they decreased and increased accompanying with disease remission and relapse. These data demonstrate that monoclonal plasma cells are related to the pathogenesis of POEMS syndrome.

Chromatin activation with H3K36me2 and compartment shift in metastatic castration-resistant prostate cancer.

Epigenetic modifiers are upregulated during the process of prostate cancer, acquiring resistance to castration therapy and becoming lethal metastatic castration-resistant prostate cancer (CRPC). However, the relationship between regulation of histone modifications and chromatin structure in CRPC has yet not fully been validated. Here, we reanalyzed publicly available clinical transcriptome and clinical outcome data and identified NSD2, a histone methyltransferase that catalyzes H3K36me2, as an epigenetic modifier that was upregulated in CRPC and whose increased expression in prostate cancer correlated with higher recurrence rate. We performed ChIP-seq, RNA-seq, and Hi-C to conduct comprehensive epigenomic and transcriptomic analyses to identify epigenetic reprogramming in CRPC. In regions where H3K36me2 was increased, H3K27me3 was decreased, and the compartment was shifted from inactive to active. In these regions, 68 aberrantly activated genes were identified as candidate downstream genes of NSD2 in CRPC. Among these genes, we identified KIF18A as critical for CRPC growth. Under NSD2 upregulation in CRPC, epigenetic alteration with H3K36me2-gain and H3K27me3-loss occurs accompanying with an inactive-to-active compartment shift, suggesting that histone modification and chromatin structure cooperatively change prostate carcinogenesis.

Retrotransposons in Werner syndrome-derived macrophages trigger type I interferon-dependent inflammation in an atherosclerosis model.

The underlying mechanisms of atherosclerosis, the second leading cause of death among Werner syndrome (WS) patients, are not fully understood. Here, we establish an in vitro co-culture system using macrophages (iMφs), vascular endothelial cells (iVECs), and vascular smooth muscle cells (iVSMCs) derived from induced pluripotent stem cells. In co-culture, WS-iMφs induces endothelial dysfunction in WS-iVECs and characteristics of the synthetic phenotype in WS-iVSMCs. Transcriptomics and open chromatin analysis reveal accelerated activation of type I interferon signaling and reduced chromatin accessibility of several transcriptional binding sites required for cellular homeostasis in WS-iMφs. Furthermore, the H3K9me3 levels show an inverse correlation with retrotransposable elements, and retrotransposable element-derived double-stranded RNA activates the DExH-box helicase 58 (DHX58)-dependent cytoplasmic RNA sensing pathway in WS-iMφs. Conversely, silencing type I interferon signaling in WS-iMφs rescues cell proliferation and suppresses cellular senescence and inflammation. These findings suggest that Mφ-specific inhibition of type I interferon signaling could be targeted to treat atherosclerosis in WS patients.

Enhancer infestation drives tumorigenic activation of inactive B compartment in Epstein-Barr virus-positive nasopharyngeal carcinoma.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated malignant epithelial tumor endemic to Southern China and Southeast Asia. While previous studies have revealed a low frequency of gene mutations in NPC, its epigenomic aberrations are not fully elucidated apart from DNA hypermethylation. Epigenomic rewiring and enhancer dysregulation, such as enhancer hijacking due to genomic structural changes or extrachromosomal DNA, drive cancer progression. METHODS: We conducted Hi-C, 4C-seq, ChIP-seq, and RNA-seq analyses to comprehensively elucidate the epigenome and interactome of NPC using C666-1 EBV(+)-NPC cell lines, NP69T immortalized nasopharyngeal epithelial cells, clinical NPC biopsy samples, and in vitro EBV infection in HK1 and NPC-TW01 EBV(-) cell lines. FINDINGS: In C666-1, the EBV genome significantly interacted with inactive B compartments of host cells; the significant association of EBV-interacting regions (EBVIRs) with B compartment was confirmed using clinical NPC and in vitro EBV infection model. EBVIRs in C666-1 showed significantly higher levels of active histone modifications compared with NP69T. Aberrant activation of EBVIRs after EBV infection was validated using in vitro EBV infection models. Within the EBVIR-overlapping topologically associating domains, 14 H3K4me3(+) genes were significantly upregulated in C666-1. Target genes of EBVIRs including PLA2G4A, PTGS2 and CITED2, interacted with the enhancers activated in EBVIRs and were highly expressed in NPC, and their knockdown significantly reduced cell proliferation. INTERPRETATION: The EBV genome contributes to NPC tumorigenesis through "enhancer infestation" by interacting with the inactive B compartments of the host genome and aberrantly activating enhancers. FUNDING: The funds are listed in the Acknowledgements section.

Integrated enhancer regulatory network by enhancer-promoter looping in gastric cancer.

Enhancer cis-regulatory elements play critical roles in gene regulation at many stages of cell growth. Enhancers in cancer cells also regulate the transcription of oncogenes. In this study, we performed a comprehensive analysis of long-range chromatin interactions, histone modifications, chromatin accessibility and expression in two gastric cancer (GC) cell lines compared to normal gastric epithelial cells. We found that GC-specific enhancers marked by histone modifications can activate a population of genes, including some oncogenes, by interacting with their proximal promoters. In addition, motif analysis of enhancer-promoter interacting enhancers showed that GC-specific transcription factors are enriched. Among them, we found that MYB is crucial for GC cell growth and activated by the enhancer with an enhancer-promoter loop and TCF7 upregulation. Clinical GC samples showed epigenetic activation of enhancers at the MYB locus and significant upregulation of TCF7 and MYB, regardless of molecular GC subtype and clinicopathological factors. Single-cell RNA sequencing of gastric mucosa with intestinal metaplasia showed high expression of TCF7 and MYB in intestinal stem cells. When we inactivated the loop-forming enhancer at the MYB locus using CRISPR interference (dCas9-KRAB), GC cell growth was significantly inhibited. In conclusion, we identified MYB as an oncogene activated by a loop-forming enhancer and contributing to GC cell growth.

Anti-proliferating and apoptosis-inducing activity of chemical compound FTI-6D in association with p53 in human cancer cell lines.

Compounds with 3,4-fused tricyclic indole (FTI) frameworks are attractive scaffolds for drug discovery. We synthesized FTI-6D, a compound with this framework, which was cytotoxic in several human cancer cell lines. FTI-6D induced apoptosis via activation of the p53 downstream mitochondria-related apoptotic pathway, characterized by an increased ratio of pro-apoptotic Bcl-2 family members to anti-apoptotic members. This change was followed by caspase-9 and caspase-3 cleavage and activation in two cancer cell lines, RKO and AGS. The anti-proliferating effect of FTI-6D was remarkably detected in eight cancer cells with wild-type TP53 (TP53_wt), including RKO and AGS, but not in seven cancer cells with mutated TP53 (TP53_mut). Additionally, p53 protein levels increased after FTI-6D treatment in TP53_wt cancer cells, and the cytotoxic effect of FTI-6D was decreased by TP53 knockdown. Accordingly, the expression of p53 downstream genes involved in apoptotic signaling pathways, such as BBC3 and TP53INP1, and those involved in cell growth inhibition, such as CDKN1A, was upregulated in TP53_wt cancer cells. These results suggest that the anti-proliferative and apoptosis-inducing activities of FTI-6D rely on p53 and the corresponding signaling processes. This study demonstrated that FTI-6D shows anti-cancer activity against TP53_wt cancer cells. FTI-6D may have potential as a prototype compound for a new drug to utilize a functional p53 pathway in TP53_wt cancer cells.