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.

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.

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.

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.

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.

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.

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.

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.

Repression of DERL3 via DNA methylation by Epstein-Barr virus latent membrane protein 1 in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is Epstein-Barr virus (EBV)-associated invasive malignancy. Increasing evidence indicates that epigenetic abnormalities, including DNA methylation, play important roles in the development of NPC. In particular, the EBV principal oncogene, latent membrane protein 1 (LMP1), is considered a key factor in inducing aberrant DNA methylation of several tumour suppressor genes in NPC, although the mechanism remains unclear. Herein, we comprehensively analysed the methylome data of Infinium BeadArray from 51 NPC and 52 normal nasopharyngeal tissues to identify LMP1-inducible methylation genes. Using hierarchical clustering analysis, we classified NPC into the high-methylation, low-methylation, and normal-like subgroups. We defined high-methylation genes as those that were methylated in the high-methylation subgroup only and common methylation genes as those that were methylated in both high- and low-methylation subgroups. Subsequently, we identified 715 LMP1-inducible methylation genes by observing the methylome data of the nasopharyngeal epithelial cell line with or without LMP1 expression. Because high-methylation genes were enriched with LMP1-inducible methylation genes, we extracted 95 high-methylation genes that overlapped with the LMP1-inducible methylation genes. Among them, we identified DERL3 as the most significantly methylated gene affected by LMP1 expression. DERL3 knockdown in cell lines resulted in significantly increased cell proliferation, migration, and invasion. Lower DERL3 expression was more frequently detected in the advanced T-stage NPC than in early T-stage NPC. These results indicate that DERL3 repression by DNA methylation contributes to NPC tumour progression.

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.

Integrated environmental, lifestyle, and epigenetic risk prediction of primary gastric neoplasia using the longitudinally monitored cohorts.

BACKGROUND: DNA methylation accumulates in non-malignant gastric mucosa after exposure to pathogens. To elucidate how environmental, methylation, and lifestyle factors interplay to influence primary gastric neoplasia (GN) risk, we analyzed longitudinally monitored cohorts in Japan and Singapore. METHODS: Asymptomatic subjects who underwent a gastric mucosal biopsy on the health check-up were enrolled. We analyzed the association between clinical factors and GN development using Cox hazard models. We further conducted comprehensive methylation analysis on selected tissues, including (i) mucosae from subjects developing GN later, (ii) mucosae from subjects not developing GN later, and (iii) GN tissues and surrounding mucosae. We also use the methylation data of mucosa collected in Singapore. The association between methylation and GN risk, as well as lifestyle and methylation, were analyzed. FINDINGS: Among 4234 subjects, GN was developed in 77 subjects. GN incidence was correlated with age, drinking, smoking, and Helicobacter pylori (HP) status. Accumulation of methylation in biopsied gastric mucosae was predictive of higher future GN risk and shorter duration to GN incidence. Whereas methylation levels were associated with HP positivity, lifestyle, and morphological alterations, DNA methylation remained an independent GN risk factor through multivariable analyses. Pro-carcinogenic epigenetic alterations initiated by HP exposure were amplified by unfavorable but modifiable lifestyle choices. Adding DNA methylation to the model with clinical factors improved the predictive ability for the GN risk. INTERPRETATION: The integration of environmental, lifestyle, and epigenetic information can provide increased resolution in the stratification of primary GN risk. FUNDING: The funds are listed in Acknowledgements section.

Proteogenomic landscape and clinical characterization of GH-producing pituitary adenomas/somatotroph pituitary neuroendocrine tumors.

The clinical characteristics of growth hormone (GH)-producing pituitary adenomas/somatotroph pituitary neuroendocrine tumors (GHomas/somatotroph PitNETs) vary across patients. In this study, we aimed to integrate the genetic alterations, protein expression profiles, transcriptomes, and clinical characteristics of GHomas/somatotroph PitNETs to identify molecules associated with acromegaly characteristics. Targeted capture sequencing and copy number analysis of 36 genes and nontargeted proteomics analysis were performed on fresh-frozen samples from 121 sporadic GHomas/somatotroph PitNETs. Targeted capture sequencing revealed GNAS as the only driver gene, as previously reported. Classification by consensus clustering using both RNA sequencing and proteomics revealed many similarities between the proteome and the transcriptome. Gene ontology analysis was performed for differentially expressed proteins between wild-type and mutant GNAS samples identified by nontargeted proteomics and involved in G protein-coupled receptor (GPCR) pathways. The results suggested that GNAS mutations impact endocrinological features in acromegaly through GPCR pathway induction. ATP2A2 and ARID5B correlated with the GH change rate in the octreotide loading test, and WWC3, SERINC1, and ZFAND3 correlated with the tumor volume change rate after somatostatin analog treatment. These results identified a biological connection between GNAS mutations and the clinical and biochemical characteristics of acromegaly, revealing molecules associated with acromegaly that may affect medical treatment efficacy.

MicroRNA-874 targets phosphomevalonate kinase and inhibits cancer cell growth via the mevalonate pathway.

The microRNA (miR) miR-874, a potential tumour suppressor, causes cell death via target gene suppression in various cancer types. Mevalonate pathway inhibition also causes cell death in breast cancer. However, the relationship between the mevalonate pathway and miR-874-induced apoptosis or its association with the tumour suppressor p53 has not been elucidated. We identified phosphomevalonate kinase (PMVK), a key mevalonate pathway enzyme, and sterol regulatory element-binding factor 2 (SREBF2), the master cholesterol biosynthesis regulator, as direct miR­874 targets. Next-generation sequencing analysis revealed a significant miR-874-mediated downregulation of PMVK and SREBF2 gene expression and p53 pathway enrichment. Luciferase reporter assays showed that miR-874 directly regulated PMVK and SREBF2. miR-874-induced apoptosis was p53 dependent, and single-cell RNA sequencing analysis demonstrated that miR-874 transfection resulted in apoptosis and p53 pathway activation. Downregulation of PMVK expression also caused cell cycle arrest and p53 pathway activation, which was rescued by geranylgeranyl pyrophosphate (GGPP) supplementation. Analysis of The Cancer Genome Atlas (TCGA) database indicated a negative correlation between miR-874 and PMVK expression and between miR-874 and SREBF2 expression. These findings suggest that miR-874 suppresses the mevalonate pathway by targeting SREBF2 and PMVK, resulting in GGPP depletion, which activates the p53 pathway and promotes cycle arrest or apoptosis.

SETD1A regulates transcriptional pause release of heme biosynthesis genes in leukemia.

Histone methyltransferase SETD1A is critical for acute myeloid leukemia (AML) cell survival, but the molecular mechanism driving SETD1A gene regulation remains elusive. To delineate the role of SETD1A, we utilize a protein degrader technology to induce rapid SETD1A degradation in AML cell lines. SETD1A degradation results in immediate downregulation of transcripts associated with DNA repair and heme biosynthesis pathways. CRISPR-based functional analyses and metabolomics reveal an essential role of SETD1A to maintain mitochondrial respiration in AML cells. These SETD1A targets are enriched in head-to-head (H2H) genes. SETD1A degradation disrupts a non-enzymatic SETD1A domain-dependent cyclin K function, increases the Ser5P RNA polymerase II (RNAPII) at the transcriptional start site (TSS), and induces the promoter-proximal pausing of RNAPII in a strand-specific manner. This study reveals a non-enzymatic role for SETD1A in transcriptional pause release and provides insight into the mechanism of RNAPII pausing and its function in cancer.

Cinobufagin inhibits proliferation of acute myeloid leukaemia cells by repressing c-Myc pathway-associated genes.

Cinobufagin is a cardiotoxic bufanolide steroid secreted by the Asiatic toad, Bufo gargarizans. Bufanolides inhibit Na+/K+ ATPase and have similar effects as cardiac glycosides, such as digitoxin or ouabain derived from toxic herbs. Recently, the anti-cancer effects of bufanolides have gained attention, however the underlying molecular mechanisms remain unclear. Selecting cinobufagin as a candidate anti-leukaemia agent, we here conducted transcriptomic analyses on the effect of cinobufagin on human acute myeloid leukaemia (AML) cell lines, HL60 and Kasumi-1. Flow cytometry analysis showed that cinobufagin induced apoptosis in both cell lines. RNA-sequencing (RNA-seq) of the two cell lines treated with cinobufagin revealed commonly downregulated genes with enrichment in the term "Myc active pathway" according to Gene Ontology (GO) analysis. Gene Set Enrichment Analysis (GSEA) of genes downregulated by cinobufagin also showed "MYC_TARGETS_V2" with the highest normalised enrichment score (NES) in both cell lines. In contrast, hallmarks such as "TNFA_SIGNALING_VIA_NFKB", "APOPTOSIS", and "TGF_BETA_SIGNALING" were significantly enriched as upregulated gene sets. Epigenetic analysis using chromatin immunoprecipitation and sequencing (ChIP-seq) confirmed that genes encoding cell death-related signalling molecules were upregulated by gain of H3K27ac, whereas downregulation of c-Myc-related genes was not accompanied by H3K27ac alteration. Cinobufagin is an anti-proliferative natural compound with c-Myc-inhibiting and epigenetic-modulating activity in acute myeloid leukaemia.

Insufficiency of non-canonical PRC1 synergizes with JAK2V617F in the development of myelofibrosis.

Insufficiency of polycomb repressive complex 2 (PRC2), which trimethylates histone H3 at lysine 27, is frequently found in primary myelofibrosis and promotes the development of JAK2V617F-induced myelofibrosis in mice by enhancing the production of dysplastic megakaryocytes. Polycomb group ring finger protein 1 (Pcgf1) is a component of PRC1.1, a non-canonical PRC1 that monoubiquitylates H2A at lysine 119 (H2AK119ub1). We herein investigated the impact of PRC1.1 insufficiency on myelofibrosis. The deletion of Pcgf1 in JAK2V617F mice strongly promoted the development of lethal myelofibrosis accompanied by a block in erythroid differentiation. Transcriptome and chromatin immunoprecipitation sequence analyses showed the de-repression of PRC1.1 target genes in Pcgf1-deficient JAK2V617F hematopoietic progenitors and revealed Hoxa cluster genes as direct targets. The deletion of Pcgf1 in JAK2V617F hematopoietic stem and progenitor cells (HSPCs), as well as the overexpression of Hoxa9, restored the attenuated proliferation of JAK2V617F progenitors. The overexpression of Hoxa9 also enhanced JAK2V617F-mediated myelofibrosis. The expression of PRC2 target genes identified in PRC2-insufficient JAK2V617F HSPCs was not largely altered in Pcgf1-deleted JAK2V617F HSPCs. The present results revealed a tumor suppressor function for PRC1.1 in myelofibrosis and suggest that PRC1.1 insufficiency has a different impact from that of PRC2 insufficiency on the pathogenesis of myelofibrosis.

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.

Identification of clonal immunoglobulin λ light-chain gene rearrangements in AL amyloidosis using next-generation sequencing.

Amyloid light-chain (AL) amyloidosis is caused by deposition of abnormally folded clonal immunoglobulin (Ig) light chains made by malignant plasma cells in the bone marrow (BM), leading to multiorgan dysfunction. However, little is known of the factors that regulate the organ tropism of amyloid deposition in this disease. We aimed to identify the clonal composition of Igλ light-chain variable region (IGLV) genes in BM cells in patients with AL amyloidosis using next-generation sequencing. Based on our definition of the clonal IGLV rearrangement (dominant clone >2.5%, dominant cluster >5%), we identified clonal IGLV in 33 of 38 patients with AL amyloidosis (86.8%), 6 of 9 with monoclonal gammopathy of undetermined significance (67%), and 7 of 7 with multiple myeloma (100%). The clones in AL amyloidosis were significantly smaller than those in multiple myeloma (p < 0.01) but comparable to those in monoclonal gammopathy of undetermined significance. Importantly, in patients with AL amyloidosis, the difference in involved and uninvolved free light chains was not correlated with the clonal size of BM plasma cells in our repertoire analysis using NGS. In summary, the clonal composition of IGLV genes in the BM was successfully identified in most patients with AL amyloidosis using NGS. The clonal size of plasma cells in the BM is small, and small malignant clones of plasma cells may secrete free light chi and cause light chain depositions in AL amyloidosis.

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 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.