Current literature suggests that epigenetically regulated super-enhancers (SEs) are drivers of aberrant gene expression in cancers. Many tumor types are still missing chromatin data to define cancer-specific SEs and their role in carcinogenesis. In this work, we develop a simple pipeline, which can utilize chromatin data from etiologically similar tumors to discover tissue-specific SEs and their target genes using gene expression and DNA methylation data. As an example, we applied our pipeline to human papillomavirus-related oropharyngeal squamous cell carcinoma (HPV + OPSCC). This tumor type is characterized by abundant gene expression changes, which cannot be explained by genetic alterations alone. Chromatin data are still limited for this disease, so we used 3627 SE elements from public domain data for closely related tissues, including normal and tumor lung, and cervical cancer cell lines. We integrated the available DNA methylation and gene expression data for HPV + OPSCC samples to filter the candidate SEs to identify functional SEs and their affected targets, which are essential for cancer development. Overall, we found 159 differentially methylated SEs, including 87 SEs that actively regulate expression of 150 nearby genes (211 SE-gene pairs) in HPV + OPSCC. Of these, 132 SE-gene pairs were validated in a related TCGA cohort. Pathway analysis revealed that the SE-regulated genes were associated with pathways known to regulate nasopharyngeal, breast, melanoma, and bladder carcinogenesis and are regulated by the epigenetic landscape in those cancers. Thus, we propose that gene expression in HPV + OPSCC may be controlled by epigenetic alterations in SE elements, which are common between related tissues. Our pipeline can utilize a diversity of data inputs and can be further adapted to SE analysis of diseased and non-diseased tissues from different organisms.

ANO1 is a calcium-activated chloride channel that is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC) and other cancers. While ANO1 expression negatively correlates with survival in several cancers, its epigenetic regulation is poorly understood. We analyzed HNSCC samples from TCGA and a separate dataset of HPV+ oropharyngeal squamous cell carcinoma (OPSCC) samples to identify differentially methylated regions. E6 and E7 transfected normal oral keratinocytes (NOK) were used to induce hypermethylation of the ANO1 promoter. We found three CpG islands that correlated with ANO1 expression, including two positively correlated with expression. Using two HNSCC datasets with differential expression of ANO1, we showed hypermethylation of positively correlated CpG islands potentiates ANO1 expression. E7 but not E6 transfection of NOK cells led to hypermethylation of a positively correlated CpG island without a change in ANO1 expression. ANO1 promoter methylation was also correlated with patient survival. Our results are the first to show the contribution of positively correlated CpG's for regulating gene expression in HNSCC. Hypermethylation of the ANO1 promoter was strongly correlated with but not sufficient to increase ANO1 expression, suggesting methylation of positively correlated CpG's likely serves as an adjunct to other mechanisms of ANO1 activation.

BACKGROUND:Targeted therapies specifically act by blocking the activity of proteins that are encoded by genes critical for tumorigenesis. However, most cancers acquire resistance and long-term disease remission is rarely observed. Understanding the time course of molecular changes responsible for the development of acquired resistance could enable optimization of patients' treatment options. Clinically, acquired therapeutic resistance can only be studied at a single time point in resistant tumors. METHODS:To determine the dynamics of these molecular changes, we obtained high throughput omics data (RNA-sequencing and DNA methylation) weekly during the development of cetuximab resistance in a head and neck cancer in vitro model. The CoGAPS unsupervised algorithm was used to determine the dynamics of the molecular changes associated with resistance during the time course of resistance development. RESULTS:CoGAPS was used to quantify the evolving transcriptional and epigenetic changes. Applying a PatternMarker statistic to the results from CoGAPS enabled novel heatmap-based visualization of the dynamics in these time course omics data. We demonstrate that transcriptional changes result from immediate therapeutic response or resistance, whereas epigenetic alterations only occur with resistance. Integrated analysis demonstrates delayed onset of changes in DNA methylation relative to transcription, suggesting that resistance is stabilized epigenetically. CONCLUSIONS:Genes with epigenetic alterations associated with resistance that have concordant expression changes are hypothesized to stabilize the resistant phenotype. These genes include FGFR1, which was associated with EGFR inhibitors resistance previously. Thus, integrated omics analysis distinguishes the timing of molecular drivers of resistance. This understanding of the time course progression of molecular changes in acquired resistance is important for the development of alternative treatment strategies that would introduce appropriate selection of new drugs to treat cancer before the resistant phenotype develops.

The incidence of HPV-related oropharyngeal squamous cell carcinoma (OPSCC) has increased more than 200% in the past 20 years. Recent genetic sequencing efforts have elucidated relevant genes in head and neck cancer, but HPV-related tumors have consistently shown few DNA mutations. In this study, we sought to analyze alternative splicing events (ASE) that could alter gene function independent of mutations. To identify ASE unique to HPV-related tumors, RNA sequencing was performed on 46 HPV-positive OPSCC and 25 normal tissue samples. A novel algorithm using outlier statistics on RNA-sequencing junction expression identified 109 splicing events, which were confirmed in a validation set from The Cancer Genome Atlas. Because the most common type of splicing event identified was an alternative start site (39%), MBD-seq genome-wide CpG methylation data were analyzed for methylation alterations at promoter regions. ASE in six genes showed significant negative correlation between promoter methylation and expression of an alternative transcriptional start site, including AKT3 The novel AKT3 transcriptional variant and methylation changes were confirmed using qRT-PCR and qMSP methods. In vitro silencing of the novel AKT3 variant resulted in significant growth inhibition of multiple head and neck cell lines, an effect not observed with wild-type AKT3 knockdown. Analysis of ASE in HPV-related OPSCC identified multiple alterations likely involved in carcinogenesis, including a novel, functionally active transcriptional variant of AKT3 Our data indicate that ASEs represent a significant mechanism of oncogenesis with untapped potential for understanding complex genetic changes that result in the development of cancer. Cancer Res; 77(19); 5248-58. ©2017 AACR.

Over a half million new cases of Head and Neck Squamous Cell Carcinoma (HNSCC) are diagnosed annually worldwide, however, 5 year overall survival is only 50% for HNSCC patients. Recently, high throughput technologies have accelerated the genome-wide characterization of HNSCC. However, comprehensive pipelines with statistical algorithms that account for HNSCC biology and perform independent confirmatory and functional validation of candidates are needed to identify the most biologically relevant genes. We applied outlier statistics to high throughput gene expression data, and identified 76 top-scoring candidates with significant differential expression in tumors compared to normal tissues. We identified 15 epigenetically regulated candidates by focusing on a subset of the genes with a negative correlation between gene expression and promoter methylation. Differential expression and methylation of 3 selected candidates (BANK1, BIN2, and DTX1) were confirmed in an independent HNSCC cohorts from Johns Hopkins and TCGA (The Cancer Genome Atlas). We further performed functional evaluation of NOTCH regulator, DTX1, which was downregulated by promoter hypermethylation in tumors, and demonstrated that decreased expression of DTX1 in HNSCC tumors maybe associated with NOTCH pathway activation and increased migration potential.

Chromatin alterations mediate mutations and gene expression changes in cancer. Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) has been utilized to study genome-wide chromatin structure in human cancer cell lines, yet numerous technical challenges limit comparable analyses in primary tumors. Here we have developed a new whole-genome analytic pipeline to optimize ChIP-Seq protocols on patient-derived xenografts from human papillomavirus-related (HPV⁺) head and neck squamous cell carcinoma (HNSCC) samples. We further associated chromatin aberrations with gene expression changes from a larger cohort of the tumor and normal samples with RNA-Seq data. We detect differential histone enrichment associated with tumor-specific gene expression variation, sites of HPV integration in the human genome, and HPV-associated histone enrichment sites upstream of cancer driver genes, which play central roles in cancer-associated pathways. These comprehensive analyses enable unprecedented characterization of the complex network of molecular changes resulting from chromatin alterations that drive HPV-related tumorigenesis. Cancer Res; 77(23); 6538-50. ©2017 AACR.

We have recently performed the characterization of alternative splicing events (ASEs) in head and neck squamous cell carcinoma, which allows dysregulation of protein expression common for cancer cells. Such analysis demonstrated a high ASE prevalence among tumor samples, including tumor-specific alternative splicing in the GSN gene.In vitro studies confirmed that overall expression of either ASE-GSN or wild-type GSN (WT-GSN) isoform inversely correlated with cell proliferation, whereas the high ratio of ASE-GSN to WT-GSN correlated with increased cellular invasion. Additionally, a change in expression of either isoform caused compensatory changes in expression of the other isoform. Our results suggest that the overall expression and the balance between GSN isoforms are mediating factors in proliferation, while increased overall expression of ASE-GSN is specific to cancer tissues. As a result, we propose ASE-GSN can serve not only as a biomarker of disease and disease progression, but also as a neoantigen for head and neck squamous cell carcinoma treatment, for which only a limited number of disease-specific targeted therapies currently exist.

It remains unclear whether causal, rather than merely correlational, relationships in molecular networks can be inferred in complex biological settings. Here we describe the HPN-DREAM network inference challenge, which focused on learning causal influences in signaling networks. We used phosphoprotein data from cancer cell lines as well as in silico data from a nonlinear dynamical model. Using the phosphoprotein data, we scored more than 2,000 networks submitted by challenge participants. The networks spanned 32 biological contexts and were scored in terms of causal validity with respect to unseen interventional data. A number of approaches were effective, and incorporating known biology was generally advantageous. Additional sub-challenges considered time-course prediction and visualization. Our results suggest that learning causal relationships may be feasible in complex settings such as disease states. Furthermore, our scoring approach provides a practical way to empirically assess inferred molecular networks in a causal sense.

Malignant pleural mesothelioma (MPM) is a highly lethal cancer of the lining of the chest cavity. To expand our understanding of MPM, we conducted a comprehensive integrated genomic study, including the most detailed analysis of BAP1 alterations to date. We identified histology-independent molecular prognostic subsets, and defined a novel genomic subtype with TP53 and SETDB1 mutations and extensive loss of heterozygosity. We also report strong expression of the immune-checkpoint gene VISTA in epithelioid MPM, strikingly higher than in other solid cancers, with implications for the immune response to MPM and for its immunotherapy. Our findings highlight new avenues for further investigation of MPM biology and novel therapeutic options. SIGNIFICANCE: Through a comprehensive integrated genomic study of 74 MPMs, we provide a deeper understanding of histology-independent determinants of aggressive behavior, define a novel genomic subtype with TP53 and SETDB1 mutations and extensive loss of heterozygosity, and discovered strong expression of the immune-checkpoint gene VISTA in epithelioid MPM.See related commentary by Aggarwal and Albelda, p. 1508.This article is highlighted in the In This Issue feature, p. 1494.

We describe a comprehensive genomic characterization of adrenocortical carcinoma (ACC). Using this dataset, we expand the catalogue of known ACC driver genes to include PRKAR1A, RPL22, TERF2, CCNE1, and NF1. Genome wide DNA copy-number analysis revealed frequent occurrence of massive DNA loss followed by whole-genome doubling (WGD), which was associated with aggressive clinical course, suggesting WGD is a hallmark of disease progression. Corroborating this hypothesis were increased TERT expression, decreased telomere length, and activation of cell-cycle programs. Integrated subtype analysis identified three ACC subtypes with distinct clinical outcome and molecular alterations which could be captured by a 68-CpG probe DNA-methylation signature, proposing a strategy for clinical stratification of patients based on molecular markers.