Spatially Resolved Niche and Tumor Microenvironmental Alterations in Gastric Cancer Peritoneal Metastases.

BACKGROUND & AIMS: Peritoneal metastasis (PM) in gastric cancer (GC) is associated with poor prognosis and significant morbidity. We sought to understand the genomic, transcriptomic, and tumor microenvironment (TME) features that contribute to peritoneal organotropism in GC. METHODS: We conducted a comprehensive multi-omic analysis of 548 samples from 326 patients, including primary tumors, matched normal tissues; peritoneal metastases, and adjacent-normal peritoneal tissues. We used whole exome sequencing, whole transcriptome sequencing, and digital spatial profiling to investigate molecular alterations, gene expression patterns, and TME characteristics associated with PM. RESULTS: Our analysis identified specific genomic alterations in primary tumors, including mutations in ELF3, CDH1, and PIGR, and TME signatures, such as stromal infiltration and M2 macrophage enrichment, associated with increased risk of PM. We observed distinct transcriptional programs and immune compositions in GCPM compared with liver metastases, highlighting the importance of the TME in transcoelomic metastasis. We found differential expression of therapeutic targets between primary tumors and PM, with lower CLDN18.2 and FGFR2b expression in PM. We unravel the roles of the TME in niche reprogramming within the peritoneum, and provide evidence of pre-metastatic niche conditioning even in early GC without clinical PM. These findings were further validated using a humanized mouse model, which demonstrated niche remodeling in the peritoneum during transcoelomic metastasis. CONCLUSION: Our study provides a comprehensive molecular characterization of GCPM and unveils key biological principles underlying transcoelomic metastasis. The identified predictive markers, therapeutic targets, and TME alterations offer potential avenues for targeted interventions and improved patient outcomes.

Regulatory enhancer profiling of mesenchymal-type gastric cancer reveals subtype-specific epigenomic landscapes and targetable vulnerabilities.

OBJECTIVE: Gastric cancer (GC) comprises multiple molecular subtypes. Recent studies have highlighted mesenchymal-subtype GC (Mes-GC) as a clinically aggressive subtype with few treatment options. Combining multiple studies, we derived and applied a consensus Mes-GC classifier to define the Mes-GC enhancer landscape revealing disease vulnerabilities. DESIGN: Transcriptomic profiles of ~1000 primary GCs and cell lines were analysed to derive a consensus Mes-GC classifier. Clinical and genomic associations were performed across >1200 patients with GC. Genome-wide epigenomic profiles (H3K27ac, H3K4me1 and assay for transposase-accessible chromatin with sequencing (ATAC-seq)) of 49 primary GCs and GC cell lines were generated to identify Mes-GC-specific enhancer landscapes. Upstream regulators and downstream targets of Mes-GC enhancers were interrogated using chromatin immunoprecipitation followed by sequencing (ChIP-seq), RNA sequencing, CRISPR/Cas9 editing, functional assays and pharmacological inhibition. RESULTS: We identified and validated a 993-gene cancer-cell intrinsic Mes-GC classifier applicable to retrospective cohorts or prospective single samples. Multicohort analysis of Mes-GCs confirmed associations with poor patient survival, therapy resistance and few targetable genomic alterations. Analysis of enhancer profiles revealed a distinctive Mes-GC epigenomic landscape, with TEAD1 as a master regulator of Mes-GC enhancers and Mes-GCs exhibiting preferential sensitivity to TEAD1 pharmacological inhibition. Analysis of Mes-GC super-enhancers also highlighted NUAK1 kinase as a downstream target, with synergistic effects observed between NUAK1 inhibition and cisplatin treatment. CONCLUSION: Our results establish a consensus Mes-GC classifier applicable to multiple transcriptomic scenarios. Mes-GCs exhibit a distinct epigenomic landscape, and TEAD1 inhibition and combinatorial NUAK1 inhibition/cisplatin may represent potential targetable options.

Comprehensive molecular phenotyping of ARID1A-deficient gastric cancer reveals pervasive epigenomic reprogramming and therapeutic opportunities.

OBJECTIVE: Gastric cancer (GC) is a leading cause of cancer mortality, with ARID1A being the second most frequently mutated driver gene in GC. We sought to decipher ARID1A-specific GC regulatory networks and examine therapeutic vulnerabilities arising from ARID1A loss. DESIGN: Genomic profiling of GC patients including a Singapore cohort (>200 patients) was performed to derive mutational signatures of ARID1A inactivation across molecular subtypes. Single-cell transcriptomic profiles of ARID1A-mutated GCs were analysed to examine tumour microenvironmental changes arising from ARID1A loss. Genome-wide ARID1A binding and chromatin profiles (H3K27ac, H3K4me3, H3K4me1, ATAC-seq) were generated to identify gastric-specific epigenetic landscapes regulated by ARID1A. Distinct cancer hallmarks of ARID1A-mutated GCs were converged at the genomic, single-cell and epigenomic level, and targeted by pharmacological inhibition. RESULTS: We observed prevalent ARID1A inactivation across GC molecular subtypes, with distinct mutational signatures and linked to a NFKB-driven proinflammatory tumour microenvironment. ARID1A-depletion caused loss of H3K27ac activation signals at ARID1A-occupied distal enhancers, but unexpectedly gain of H3K27ac at ARID1A-occupied promoters in genes such as NFKB1 and NFKB2. Promoter activation in ARID1A-mutated GCs was associated with enhanced gene expression, increased BRD4 binding, and reduced HDAC1 and CTCF occupancy. Combined targeting of promoter activation and tumour inflammation via bromodomain and NFKB inhibitors confirmed therapeutic synergy specific to ARID1A-genomic status. CONCLUSION: Our results suggest a therapeutic strategy for ARID1A-mutated GCs targeting both tumour-intrinsic (BRD4-assocatiated promoter activation) and extrinsic (NFKB immunomodulation) cancer phenotypes.

Epigenetic promoter alterations in GI tumour immune-editing and resistance to immune checkpoint inhibition.

OBJECTIVES: Epigenomic alterations in cancer interact with the immune microenvironment to dictate tumour evolution and therapeutic response. We aimed to study the regulation of the tumour immune microenvironment through epigenetic alternate promoter use in gastric cancer and to expand our findings to other gastrointestinal tumours. DESIGN: Alternate promoter burden (APB) was quantified using a novel bioinformatic algorithm (proActiv) to infer promoter activity from short-read RNA sequencing and samples categorised into APBhigh, APBint and APBlow. Single-cell RNA sequencing was performed to analyse the intratumour immune microenvironment. A humanised mouse cancer in vivo model was used to explore dynamic temporal interactions between tumour kinetics, alternate promoter usage and the human immune system. Multiple cohorts of gastrointestinal tumours treated with immunotherapy were assessed for correlation between APB and treatment outcomes. RESULTS: APBhigh gastric cancer tumours expressed decreased levels of T-cell cytolytic activity and exhibited signatures of immune depletion. Single-cell RNAsequencing analysis confirmed distinct immunological populations and lower T-cell proportions in APBhigh tumours. Functional in vivo studies using 'humanised mice' harbouring an active human immune system revealed distinct temporal relationships between APB and tumour growth, with APBhigh tumours having almost no human T-cell infiltration. Analysis of immunotherapy-treated patients with GI cancer confirmed resistance of APBhigh tumours to immune checkpoint inhibition. APBhigh gastric cancer exhibited significantly poorer progression-free survival compared with APBlow (median 55 days vs 121 days, HR 0.40, 95% CI 0.18 to 0.93, p=0.032). CONCLUSION: These findings demonstrate an association between alternate promoter use and the tumour microenvironment, leading to immune evasion and immunotherapy resistance.

HNF4α pathway mapping identifies wild-type IDH1 as a targetable metabolic node in gastric cancer.

OBJECTIVE: Gastric cancer (GC) is a leading cause of cancer mortality. Previous studies have shown that hepatocyte nuclear factor-4α (HNF4α) is specifically overexpressed in GC and functionally required for GC development. In this study, we investigated, on a genome-wide scale, target genes of HNF4α and oncogenic pathways driven by HNF4α and HNF4α target genes. DESIGN: We performed HNF4α chromatin immunoprecipitation followed by sequencing across multiple GC cell lines, integrating HNF4α occupancy data with (epi)genomic and transcriptome data of primary GCs to define HNF4α target genes of in vitro and in vivo relevance. To investigate mechanistic roles of HNF4α and HNF4α targets, we performed cancer metabolic measurements, drug treatments and functional assays including murine xenograft experiments. RESULTS: Gene expression analysis across 19 tumour types revealed HNF4α to be specifically upregulated in GCs. Unbiased pathway analysis revealed organic acid metabolism as the top HNF4α-regulated pathway, orthogonally supported by metabolomic analysis. Isocitrate dehydrogenase 1 (IDH1) emerged as a convergent HNF4α direct target gene regulating GC metabolism. We show that wild-type IDH1 is essential for GC cell survival, and that certain GC cells can be targeted by IDH1 inhibitors. CONCLUSIONS: Our results highlight a role for HNF4α in sustaining GC oncogenic metabolism, through the regulation of IDH1. Drugs targeting wild-type IDH1 may thus have clinical utility in GCs exhibiting HNF4α overexpression, expanding the role of IDH1 in cancer beyond IDH1/2 mutated malignancies.

Integrated paired-end enhancer profiling and whole-genome sequencing reveals recurrent CCNE1 and IGF2 enhancer hijacking in primary gastric adenocarcinoma.

OBJECTIVE: Genomic structural variations (SVs) causing rewiring of cis-regulatory elements remain largely unexplored in gastric cancer (GC). To identify SVs affecting enhancer elements in GC (enhancer-based SVs), we integrated epigenomic enhancer profiles revealed by paired-end H3K27ac ChIP-sequencing from primary GCs with tumour whole-genome sequencing (WGS) data (PeNChIP-seq/WGS). DESIGN: We applied PeNChIP-seq to 11 primary GCs and matched normal tissues combined with WGS profiles of >200 GCs. Epigenome profiles were analysed alongside matched RNA-seq data to identify tumour-associated enhancer-based SVs with altered cancer transcription. Functional validation of candidate enhancer-based SVs was performed using CRISPR/Cas9 genome editing, chromosome conformation capture assays (4C-seq, Capture-C) and Hi-C analysis of primary GCs. RESULTS: PeNChIP-seq/WGS revealed ~150 enhancer-based SVs in GC. The majority (63%) of SVs linked to target gene deregulation were associated with increased tumour expression. Enhancer-based SVs targeting CCNE1, a key driver of therapy resistance, occurred in 8% of patients frequently juxtaposing diverse distal enhancers to CCNE1 proximal regions. CCNE1-rearranged GCs were associated with high CCNE1 expression, disrupted CCNE1 topologically associating domain (TAD) boundaries, and novel TAD interactions in CCNE1-rearranged primary tumours. We also observed IGF2 enhancer-based SVs, previously noted in colorectal cancer, highlighting a common non-coding genetic driver alteration in gastric and colorectal malignancies. CONCLUSION: Integrated paired-end NanoChIP-seq and WGS of gastric tumours reveals tumour-associated regulatory SV in regions associated with both simple and complex genomic rearrangements. Genomic rearrangements may thus exploit enhancer-hijacking as a common mechanism to drive oncogene expression in GC.

Spatiotemporal genomic profiling of intestinal metaplasia reveals clonal dynamics of gastric cancer progression.

Intestinal metaplasia (IM) is a pre-malignant condition of the gastric mucosa associated with increased gastric cancer (GC) risk. Analyzing 1,256 gastric samples (1,152 IMs) across 692 subjects from a prospective 10-year study, we identify 26 IM driver genes in diverse pathways including chromatin regulation (ARID1A) and intestinal homeostasis (SOX9). Single-cell and spatial profiles highlight changes in tissue ecology and IM lineage heterogeneity, including an intestinal stem-cell dominant cellular compartment linked to early malignancy. Expanded transcriptome profiling reveals expression-based molecular subtypes of IM associated with incomplete histology, antral/intestinal cell types, ARID1A mutations, inflammation, and microbial communities normally associated with the healthy oral tract. We demonstrate that combined clinical-genomic models outperform clinical-only models in predicting IMs likely to transform to GC. By highlighting strategies for accurately identifying IM patients at high GC risk and a role for microbial dysbiosis in IM progression, our results raise opportunities for GC precision prevention and interception.

Chromatin Rewiring by Mismatch Repair Protein MSH2 Alters Cell Adhesion Pathways and Sensitivity to BET Inhibition in Gastric Cancer.

Mutations in the DNA mismatch repair gene MSH2 are causative of microsatellite instability (MSI) in multiple cancers. Here, we discovered that besides its well-established role in DNA repair, MSH2 exerts a novel epigenomic function in gastric cancer. Unbiased CRISPR-based mass spectrometry combined with genome-wide CRISPR functional screening revealed that in early-stage gastric cancer MSH2 genomic binding is not randomly distributed but rather is associated specifically with tumor-associated super-enhancers controlling the expression of cell adhesion genes. At these loci, MSH2 genomic binding was required for chromatin rewiring, de novo enhancer-promoter interactions, maintenance of histone acetylation levels, and regulation of cell adhesion pathway expression. The chromatin function of MSH2 was independent of its DNA repair catalytic activity but required MSH6, another DNA repair gene, and recruitment to gene loci by the SWI/SNF chromatin remodeler SMARCA4/BRG1. Loss of MSH2 in advanced gastric cancers was accompanied by deficient cell adhesion pathway expression, epithelial-mesenchymal transition, and enhanced tumorigenesis in vitro and in vivo. However, MSH2-deficient gastric cancers also displayed addiction to BAZ1B, a bromodomain-containing family member, and consequent synthetic lethality to bromodomain and extraterminal motif (BET) inhibition. Our results reveal a role for MSH2 in gastric cancer epigenomic regulation and identify BET inhibition as a potential therapy in MSH2-deficient gastric malignancies. SIGNIFICANCE: DNA repair protein MSH2 binds and regulates cell adhesion genes by enabling enhancer-promoter interactions, and loss of MSH2 causes deficient cell adhesion and bromodomain and extraterminal motif inhibitor synthetic lethality in gastric cancer.

"3G" Trial: An RNA Editing Signature to Guide Gastric Cancer Chemotherapy.

Gastric cancer cases are often diagnosed at an advanced stage with poor prognosis. Platinum-based chemotherapy has been internationally accepted as first-line therapy for inoperable or metastatic gastric cancer. To achieve greater benefits, selection of patients eligible for this treatment is critical. Although gene expression profiling has been widely used as a genomic classifier to identify molecular subtypes of gastric cancer and to stratify patients for different chemotherapy regimens, its prediction accuracy can be improved. Adenosine-to-inosine (A-to-I) RNA editing has emerged as a new player contributing to gastric cancer development and progression, offering potential clinical utility for diagnosis and treatment. Using a systematic computational approach followed by both in vitro validations and in silico validations in The Cancer Genome Atlas (TCGA), we conducted a transcriptome-wide RNA editing analysis of a cohort of 104 patients with advanced gastric cancer and identified an RNA editing (GCRE) signature to guide gastric cancer chemotherapy. RNA editing events stood as a prognostic and predictive biomarker in advanced gastric cancer. A GCRE score based on the GCRE signature consisted of 50 editing sites associated with 29 genes, predicting response to chemotherapy with a high accuracy (84%). Of note, patients demonstrating higher editing levels of this panel of sites presented a better overall response. Consistently, gastric cancer cell lines with higher editing levels showed higher chemosensitivity. Applying the GCRE score on TCGA dataset confirmed that responders had significantly higher levels of editing in advanced gastric cancer. Overall, this newly defined GCRE signature reliably stratifies patients with advanced gastric cancer and predicts response from chemotherapy. SIGNIFICANCE: This study describes a novel A-to-I RNA editing signature as a prognostic and predictive biomarker in advanced gastric cancer, providing a new tool to improve patient stratification and response to therapy.

Integrative epigenomic and high-throughput functional enhancer profiling reveals determinants of enhancer heterogeneity in gastric cancer.

BACKGROUND: Enhancers are distal cis-regulatory elements required for cell-specific gene expression and cell fate determination. In cancer, enhancer variation has been proposed as a major cause of inter-patient heterogeneity-however, most predicted enhancer regions remain to be functionally tested. METHODS: We analyzed 132 epigenomic histone modification profiles of 18 primary gastric cancer (GC) samples, 18 normal gastric tissues, and 28 GC cell lines using Nano-ChIP-seq technology. We applied Capture-based Self-Transcribing Active Regulatory Region sequencing (CapSTARR-seq) to assess functional enhancer activity. An Activity-by-contact (ABC) model was employed to explore the effects of histone acetylation and CapSTARR-seq levels on enhancer-promoter interactions. RESULTS: We report a comprehensive catalog of 75,730 recurrent predicted enhancers, the majority of which are GC-associated in vivo (> 50,000) and associated with lower somatic mutation rates inferred by whole-genome sequencing. Applying CapSTARR-seq to the enhancer catalog, we observed significant correlations between CapSTARR-seq functional activity and H3K27ac/H3K4me1 levels. Super-enhancer regions exhibited increased CapSTARR-seq signals compared to regular enhancers, even when decoupled from native chromatin contexture. We show that combining histone modification and CapSTARR-seq functional enhancer data improves the prediction of enhancer-promoter interactions and pinpointing of germline single nucleotide polymorphisms (SNPs), somatic copy number alterations (SCNAs), and trans-acting TFs involved in GC expression. We identified cancer-relevant genes (ING1, ARL4C) whose expression between patients is influenced by enhancer differences in genomic copy number and germline SNPs, and HNF4α as a master trans-acting factor associated with GC enhancer heterogeneity. CONCLUSIONS: Our results indicate that combining histone modification and functional assay data may provide a more accurate metric to assess enhancer activity than either platform individually, providing insights into the relative contribution of genetic (cis) and regulatory (trans) mechanisms to GC enhancer functional heterogeneity.

Cross-species chromatin interactions drive transcriptional rewiring in Epstein-Barr virus-positive gastric adenocarcinoma.

Epstein-Barr virus (EBV) is associated with several human malignancies including 8-10% of gastric cancers (GCs). Genome-wide analysis of 3D chromatin topologies across GC lines, primary tissue and normal gastric samples revealed chromatin domains specific to EBV-positive GC, exhibiting heterochromatin-to-euchromatin transitions and long-range human-viral interactions with non-integrated EBV episomes. EBV infection in vitro suffices to remodel chromatin topology and function at EBV-interacting host genomic loci, converting H3K9me3+ heterochromatin to H3K4me1+/H3K27ac+ bivalency and unleashing latent enhancers to engage and activate nearby GC-related genes (for example TGFBR2 and MZT1). Higher-order epigenotypes of EBV-positive GC thus signify a novel oncogenic paradigm whereby non-integrative viral genomes can directly alter host epigenetic landscapes ('enhancer infestation'), facilitating proto-oncogene activation and tumorigenesis.

Genomic and epigenomic EBF1 alterations modulate TERT expression in gastric cancer.

Transcriptional reactivation of telomerase catalytic subunit (TERT) is a frequent hallmark of cancer, occurring in 90% of human malignancies. However, specific mechanisms driving TERT reactivation remain obscure for many tumor types and in particular gastric cancer (GC), a leading cause of global cancer mortality. Here, through comprehensive genomic and epigenomic analysis of primary GCs and GC cell lines, we identified the transcription factor early B cell factor 1 (EBF1) as a TERT transcriptional repressor and inactivation of EBF1 function as a major cause of TERT upregulation. Abolishment of EBF1 function occurs through 3 distinct (epi)genomic mechanisms. First, EBF1 is epigenetically silenced via DNA methyltransferase, polycomb-repressive complex 2 (PRC2), and histone deacetylase activity in GCs. Second, recurrent, somatic, and heterozygous EBF1 DNA-binding domain mutations result in the production of dominant-negative EBF1 isoforms. Third, more rarely, genomic deletions and rearrangements proximal to the TERT promoter remobilize or abolish EBF1-binding sites, derepressing TERT and leading to high TERT expression. EBF1 is also functionally required for various malignant phenotypes in vitro and in vivo, highlighting its importance for GC development. These results indicate that multimodal genomic and epigenomic alterations underpin TERT reactivation in GC, converging on transcriptional repressors such as EBF1.

Epigenomic Promoter Alterations Amplify Gene Isoform and Immunogenic Diversity in Gastric Adenocarcinoma.

Promoter elements play important roles in isoform and cell type-specific expression. We surveyed the epigenomic promoter landscape of gastric adenocarcinoma, analyzing 110 chromatin profiles (H3K4me3, H3K4me1, H3K27ac) of primary gastric cancers, gastric cancer lines, and nonmalignant gastric tissues. We identified nearly 2,000 promoter alterations (somatic promoters), many deregulated in various epithelial malignancies and mapping frequently to alternative promoters within the same gene, generating potential pro-oncogenic isoforms (RASA3). Somatic promoter-associated N-terminal peptides displaying relative depletion in tumors exhibited high-affinity MHC binding predictions and elicited potent T-cell responses in vitro, suggesting a mechanism for reducing tumor antigenicity. In multiple patient cohorts, gastric cancers with high somatic promoter usage also displayed reduced T-cell cytolytic marker expression. Somatic promoters are enriched in PRC2 occupancy, display sensitivity to EZH2 therapeutic inhibition, and are associated with novel cancer-associated transcripts. By generating tumor-specific isoforms and decreasing tumor antigenicity, epigenomic promoter alterations may thus drive intrinsic tumorigenesis and also allow nascent cancers to evade host immunity.Significance: We apply epigenomic profiling to demarcate the promoter landscape of gastric cancer. Many tumor-specific promoters activate different promoters in the same gene, some generating pro-oncogenic isoforms. Tumor-specific promoters also reduce tumor antigenicity by causing relative depletion of immunogenic peptides, contributing to cancer immunoediting and allowing tumors to evade host immune attack. Cancer Discov; 7(6); 630-51. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 539.