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Purpose:Investigation of biological mechanisms underlying genetic alterations in cancer can assist the understanding of etiology and identify the potential prognostic biomarkers.Experimental Design:We performed an integrative genomic analysis for a total of 731 nasopharyngeal carcinoma cases from five independent nasopharyngeal carcinoma cohorts to identify the genetic events associated with clinical outcomes.Results:In addition to the known mutational signatures associated with aging, APOBEC and mismatch repair (MMR), a new signature for homologous recombination deficiency (BRCAness) was discovered in 64 of 216 (29.6%) cases in the discovery set including three cohorts. This signature appeared more frequently in the recurrent and metastatic tumors and significantly correlated with shorter overall survival (OS) in the primary tumors. Independent prognostic value of MMR and BRCAness signatures was revealed by multivariable Cox analysis after adjustment for clinical parameters and stratification by studies. The cases with both signatures had much worse clinical outcome than those without these signatures [hazard ratio (HR), 12.4; P = 0.002]. This correlation was confirmed in the validation set (HR, 8.9; P = 0.003). The BRCAness signature is highly associated with BRCA2 pathogenic germline or somatic alterations (7.8% vs. 0%; P = 0.002). Targeted sequencing results from a prospective nasopharyngeal carcinoma cohort (N = 402) showed that the cases carrying BRCA2 germline rare variants are more likely to have poor OS and progression-free survival.Conclusions:Our study highlights importance of defects of DNA repair machinery in nasopharyngeal carcinoma pathogenesis and their prognostic values for clinical implications. These signatures will be useful for patient stratification to evaluate conventional and new treatment for precision medicine in nasopharyngeal carcinoma.

Figure S5: Methylation of selected genes between NPC and adjacent non-tumor tissues.

Supplementary Legend

Table S1: Clinical parameters for the HK-3 cohort for targeted sequencing (N=402);Table S2: Sample size estimation and power calculation for study cohorts;Table S3: Mutational signatures and clinical parameters in primary NPC (N=178);Table S4: All the pathogenic germline and somatic alterations at BRCA2 (NM_000059) detected in NPC (N=216);Table S5: Germline and somatic alterations at the selected DNA damage and repair genes in 216 NPC cases for the genes with differential expression and SCNAs in NPC;Table S6: Germline rare variants detected at BRCA2 (NM_000059) in the HK-3 cohort (N=402);Table S7: Association of BRCA2 germline rare variants with clinical parameters in the HK-3 cohort (N=402);Table S8: Differentially methylated CpG sites between signature 3-positive and -negative cases

Background: Epstein-Barr virus (EBV) can be detected in more than 90% of NPC patients in Hong Kong. It has been shown that EBV infection could hijack the human epigenome in B cell lymphoma and EBV+ gastric cancer, however, the role of EBV in epigenetic dysregulation remains largely unknown in NPC. Recently, our epigenomic landscape study demonstrated EBV was associated with changes of chromatin accessibility in NPC, which were largely involved in the binding sites of CTCF, a master regulator of chromosome organization. Therefore, we aim to further characterize the 3D chromatin structure in NPC. Methods: We used the Hi-C (Omni-C Seq) to profile the chromatin interactions in the EBV-positive (EBV+) NPC, EBV-negative (EBV-) NPC, and normal immortalized epithelial (NPE) cell lines. The Omni-C Seq data were integrated with the ATAC-Seq, RNA-Seq, and CUT&RUN-Seq data. The histone markers including H3K4me3, H3K27ac, and H3K27me3, were applied to classify the active and inactive status of the chromatins. The chromatin looping and topologically associated domains (TADs) were defined by the CTCF binding sites. Results: We observed a much larger compartmentalization size in EBV+ NPC with a consistent increase of active to inactive (A to B) compartments, compared to NPE and EBV- NPC. Pathway enrichment analysis showed that these changes potentially influenced immune response, cell differentiation and development, metabolism, ERBB pathway, and calcium signaling transduction. In addition to the changes in compartmentalization, we observed a reduction of CTCF binding signals at the TADs boundaries and the loss of chromatin looping at the 10kb resolution. Consistently, the aggregate peak analysis has shown a significant reduction of the CTCF anchor, indicating the abandonment of architectural CTCF in EBV+ NPC. More importantly, we found EBV tethering to the host chromatins. In the EBV genome, the tethering sites were often located at the regions associated with OriP, EBNA1, and BARTs. In the human genome, the tethering sites were dramatically enriched in the inactive to active (B to A) compartments, which were highly associated with the enhancer regions with H3K27ac. We identified a specific target gene CD74 with EBV tethering at the nearby super-enhancer region. Our results suggest the upregulation of CD74 could be controlled via remodulating the host 3D genome in EBV+ NPC. The spatial transcriptome analysis indicated that the expression of CD74 was highly correlated with HLA-DRA. The spatial analysis further showed that the NPC patients with upregulated HLA-DRA often had de novo distant metastasis after treatment (p=0.007). Conclusions: The EBV tethering to the host genome has a significant impact on host 3D genome organization, affecting chromatin accessibility and gene regulation, providing survival advantages of cancer cell growth and potentially contributing to immune suppression and metastasis. Citation Format: Dittman Lai-Shun Chung, Larry Ka-Yue Chow, Wei Dai. Characterization of 3D genome in nasopharyngeal carcinoma (NPC). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4716.

Distant metastasis remains the leading cause of treatment failure in patients with nasopharyngeal carcinoma (NPC), making it critical to identify efficient therapeutic targets for metastatic NPC. Previous studies have demonstrated that deoxynucleotidyltransferase terminal-interacting protein 1 (DNTTIP1) is associated with the development of various types of cancer. However, its role and mechanism in NPC have not been explored.RNA-seq profiling was performed for three pairs of NPC and normal nasopharynx tissues. DNTTIP1 expression in NPC specimens was detected by immunohistochemistry. In vitro and in vivo assays were used to investigate the function of DNTTIP1. The molecular mechanism was determined using RT-qPCR, western blotting, RNA-seq, luciferase reporter assays, ChIP assays, and co-IP assays.DNTTIP1 was found to be significantly upregulated in NPC tissues. Furthermore, DNTTIP1 promoted NPC growth and metastasis in vitro and in vivo. Upregulation of DNTTIP1 in NPC indicated poor clinical outcomes. Mechanistically, DNTTIP1 suppressed DUSP2 gene expression via recruiting HDAC1 to its promoter and maintaining a deacetylated state of histone H3K27. The downregulation of DUSP2 resulted in aberrant activation of the ERK signaling and elevated MMP2 levels, promoting NPC metastasis. Chidamide, an HDAC inhibitor, was shown to suppress NPC metastasis by regulating the DNTTIP1/HDAC1-DUSP2 axis.Our findings demonstrate that DNTTIP1 not only regulates NPC metastasis but also independently predicts NPC prognosis. Furthermore, targeting DNTTIP1/HDAC1 by Chidamide may benefit NPC patients with metastasis.This work was supported by the National Natural Science Foundation of China (No. 81872464, 82073243).

The tumor microenvironment (TME) of nasopharyngeal carcinoma (NPC) harbors a heterogeneous and dynamic stromal population. A comprehensive understanding of this tumor-specific ecosystem is necessary to enhance cancer diagnosis, therapeutics, and prognosis. However, recent advances based on bulk RNA sequencing remain insufficient to construct an in-depth landscape of infiltrating stromal cells in NPC. Here we apply single-cell RNA sequencing to 66,627 cells from 14 patients, integrated with clonotype identification on T and B cells. We identify and characterize five major stromal clusters and 36 distinct subpopulations based on genetic profiling. By comparing with the infiltrating cells in the non-malignant microenvironment, we report highly representative features in the TME, including phenotypic abundance, genetic alternations, immune dynamics, clonal expansion, developmental trajectory, and molecular interactions that profoundly influence patient prognosis and therapeutic outcome. The key findings are further independently validated in two single-cell RNA sequencing cohorts and two bulk RNA-sequencing cohorts. In the present study, we reveal the correlation between NPC-specific characteristics and progression-free survival. Together, these data facilitate the understanding of the stromal landscape and immune dynamics in NPC patients and provides deeper insights into the development of prognostic biomarkers and therapeutic targets in the TME., The tumor microenvironment can influence patient survival response to therapy. Here, the authors used single-cell sequencing to investigate the microenvironment of nasopharyngeal cancer and identify tumor-specific signatures in five stromal clusters of cells that may influence patient survival.

Purpose: Investigation of biological mechanisms underlying genetic alterations in cancer can assist the understanding of etiology and identify the potential prognostic biomarkers. Experimental Design: We performed an integrative genomic analysis for a total of 731 nasopharyngeal carcinoma cases from five independent nasopharyngeal carcinoma cohorts to identify the genetic events associated with clinical outcomes. Results: In addition to the known mutational signatures associated with aging, APOBEC and mismatch repair (MMR), a new signature for homologous recombination deficiency (BRCAness) was discovered in 64 of 216 (29.6%) cases in the discovery set including three cohorts. This signature appeared more frequently in the recurrent and metastatic tumors and significantly correlated with shorter overall survival (OS) in the primary tumors. Independent prognostic value of MMR and BRCAness signatures was revealed by multivariable Cox analysis after adjustment for clinical parameters and stratification by studies. The cases with both signatures had much worse clinical outcome than those without these signatures [hazard ratio (HR), 12.4; P = 0.002]. This correlation was confirmed in the validation set (HR, 8.9; P = 0.003). The BRCAness signature is highly associated with BRCA2 pathogenic germline or somatic alterations (7.8% vs. 0%; P = 0.002). Targeted sequencing results from a prospective nasopharyngeal carcinoma cohort (N = 402) showed that the cases carrying BRCA2 germline rare variants are more likely to have poor OS and progression-free survival. Conclusions: Our study highlights importance of defects of DNA repair machinery in nasopharyngeal carcinoma pathogenesis and their prognostic values for clinical implications. These signatures will be useful for patient stratification to evaluate conventional and new treatment for precision medicine in nasopharyngeal carcinoma.

Chromatin regulators (CRs) can dynamically modulate chromatin architecture to epigenetically regulate gene expression in response to intrinsic and extrinsic signalling cues. Somatic alterations or misexpression of CRs might reprogram the epigenomic landscape of chromatin, which in turn lead to a wide range of common diseases, notably cancer. Here, we present CR2Cancer, a comprehensive annotation and visualization database for CRs in human cancer constructed by high throughput data analysis and literature mining. We collected and integrated genomic, transcriptomic, proteomic, clinical and functional information for over 400 CRs across multiple cancer types. We also built diverse types of CR-associated relations, including cancer type dependent (CR-target and miRNA-CR) and independent (protein-protein interaction and drug-target) ones. Furthermore, we manually curated around 6000 items of aberrant molecular alterations and interactions of CRs in cancer development from 5007 publications. CR2Cancer provides a user-friendly web interface to conveniently browse, search and download data of interest. We believe that this database would become a valuable resource for cancer epigenetics investigation and potential clinical application. CR2Cancer is freely available at http://cis.hku.hk/CR2Cancer.

Background: Investigation of biological mechanisms underlying genetic alterations in cancer can assist the understanding of the inter-patient heterogeneity and identify new therapeutic targets. Although multiple genomic studies have been carried out in nasopharyngeal carcinoma (NPC), the detailed mechanisms underlying these genetic alterations and NPC pathogenesis remain unclear. Aims: We aim to identify the additional genetic events driving tumorigenesis by increasing the statistical power to distinguish the infrequent driver events from passengers, gain insights in the mechanisms underlying genetic alterations and evaluate their prognostic value for overall survival. Our ultimate goal is to identify potential new therapeutic targets for NPC treatment. Methods: We performed an integrative genomic and transcriptomic analysis for a total of 216 NPC cases from three genomics studies to catalogue the somatic mutational signatures, somatic copy number alterations (SCNAs) and gene expression changes in NPC. Results: Three clusters with distinct SCNA patterns were uncovered. Cluster 1 (SCNA-L-gain), accounting for 66.7% of total cases, has consistent CN loss in chromosomes 3, 14 and 16, which are typical SCNAs in NPC; Cluster 2 (SCNA-M-gain, 8.2%) is characterized as a moderate level of CN gain involved in selected chromosomes 17, 19 and 22. Strikingly, Cluster 3 (SCNA-H-gain, 25.1%) has extensive CN gains across the genome with regional loss near telomeres in several chromosomes. Noticeably, a new mutational signature relevant to the deficiency of the homologous recombination (HR) repair pathway (BRCAness) was discovered in a subset of NPC cases (29.6%). The survival analysis indicates that this mutational signature is significantly associated with shorter overall survival after adjustment for stage, gender and age and stratification by studies (P=0.018, HR=2.2, 95% CI 1.1-4.4). All the cases harboring the pathogenic BRCA2 germline and somatic mutations carry this mutational signature, but only account for 7.8% of such cases. A strong BRCAness signature is more frequently found in the SCNA-H-gain cluster (P=0.012), indicating HR deficiency may play a role for genomic instability in this subset of NPC patients. Conclusions: In NPC, there is a molecular subtype with extensively high copy number gain and a strong BRCAness signature. Discovery of this molecular subtype supports prospective clinical trials investigating agents, such as PARP inhibitor, to target this subset of patients. Acknowledgements: This study was funded by the Hong Kong Research Grants Council grant (AoE/M-06/08) to MLL Citation Format: Wei Dai, Dittman Lai-Shun Chung, Larry Ka-Yue Chow, Merrin Man-Long Leong, Candy King-Chi Chan, Josephine Mun-Yee Ko, Maria Li Lung. Nasopharyngeal carcinoma molecular subgroups for somatic copy number alterations (SCNAs) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4734.