Head and Neck Squamous Cell Carcinoma (HNSCC) is a heterogeneous disease of significant mortality and with limited treatment options. Recent genomic analysis of HNSCC tumors has identified several distinct molecular classes, of which the mesenchymal subtype is associated with Epithelial to Mesenchymal Transition (EMT) and shown to correlate with poor survival and drug resistance. Here, we utilize an integrated approach to characterize the molecular function of ETS1, an oncogenic transcription factor specifically enriched in Mesenchymal tumors. To identify the global ETS1 cistrome, we have performed integrated analysis of RNA-Seq, ChIP-Seq and epigenomic datasets in SCC25, a representative ETS1 high mesenchymal HNSCC cell line. Our studies implicate ETS1 as a crucial regulator of broader oncogenic processes and specifically Mesenchymal phenotypes, such as EMT and cellular invasion. We found that ETS1 preferentially binds cancer specific regulator elements, in particular Super-Enhancers of key EMT genes, highlighting its role as a master regulator. Finally, we show evidence that ETS1 plays a crucial role in regulating the TGF-β pathway in Mesenchymal cell lines and in leading-edge cells in primary HNSCC tumors that are endowed with partial-EMT features. Collectively our study highlights ETS1 as a key regulator of TGF-β associated EMT and reveals new avenues for sub-type specific therapeutic intervention.

Head and Neck Squamous Cell Carcinoma (HNSCC) is a heterogeneous disease with relatively high morbidity and mortality rates. The lack of effective therapies, high recurrence rates and drug resistance driven in part, by tumor heterogeneity, contribute to the poor prognosis for patients diagnosed with this cancer. This problem is further exacerbated by the fact that key regulatory factors contributing to the disease diversity remains largely elusive. Here, we have identified EHF as an important member of the ETS family of transcription factors that is highly expressed in normal oral tissues, but lost during HNSCC progression. Interestingly, HNSCC tumors and cell lines exhibited a dichotomy of high and low EHF expression, and patients whose tumors retained EHF expression showed significantly better prognosis, suggesting a potential tumor suppressive role for EHF. To address this, we have performed gain and loss of function studies and leveraged bulk and single-cell cancer genomic datasets to identify global EHF targets by RNA-sequencing (RNA-seq) and Chromatin Immunoprecipitation and next generation sequencing (ChIP-seq) experiments of HNSCC cell lines. These mechanistic studies have revealed that EHF, acts as a regulator of a broad spectrum of metabolic processes, specifically targeting regulators of redox homeostasis such as NRF2 and SOX2. Our immunostaining results confirm the mutually exclusive expression patterns of EHF and SOX2 in HNSCC tumors and suggest a possible role for these two factors in establishing discrete metabolic states within the tumor microenvironment. Taken together, EHF may serve as a novel prognostic marker for classifying HNSCC patients for actionable and targeted therapeutic intervention.

The complex heterogeneity of head and neck squamous cell carcinoma (HNSCC) reflects a diverse underlying etiology. This heterogeneity is also apparent within Human Papillomavirus-positive (HPV+) HNSCC subtypes, which have distinct gene expression profiles and patient outcomes. One aggressive HPV+ HNSCC subtype is characterized by elevated expression of genes involved in keratinization, a process regulated by the oncogenic transcription factor ΔNp63. Furthermore, the human TP63 gene locus is a frequent HPV integration site and HPV oncoproteins drive ΔNp63 expression, suggesting an unexplored functional link between ΔNp63 and HPV+ HNSCC. Here we show that HPV+ HNSCCs can be molecularly stratified according to ΔNp63 expression levels and derive a ΔNp63-associated gene signature profile for such tumors. We leveraged RNA-seq data from p63 knockdown cells and ChIP-seq data for p63 and histone marks from two ΔNp63high HPV+ HNSCC cell lines to identify an epigenetically refined ΔNp63 cistrome. Our integrated analyses reveal crucial ΔNp63-bound super-enhancers likely to mediate HPV+ HNSCC subtype-specific gene expression that is anchored, in part, by the PI3K-mTOR pathway. These findings implicate ΔNp63 as a key regulator of essential oncogenic pathways in a subtype of HPV+ HNSCC that can be exploited as a biomarker for patient stratification and treatment choices.

Oral squamous cell carcinoma (OSCC) is the most common malignancy of the oral cavity and is linked to tobacco exposure, alcohol consumption, and human papillomavirus infection. Despite therapeutic advances, a lack of molecular understanding of disease etiology, and delayed diagnoses continue to negatively affect survival. The identification of oncogenic drivers and prognostic biomarkers by leveraging bulk and single-cell RNA-sequencing datasets of OSCC can lead to more targeted therapies and improved patient outcomes. However, the generation, analysis, and continued utilization of additional genetic and genomic tools are warranted. Tobacco-induced OSCC can be modeled in mice via 4-nitroquinoline 1-oxide (4NQO), which generates a spectrum of neoplastic lesions mimicking human OSCC and upregulates the oncogenic master transcription factor p63. Here, we molecularly characterized established mouse 4NQO treatment-derived OSCC cell lines and utilized RNA and chromatin immunoprecipitation-sequencing to uncover the global p63 gene regulatory and signaling network. We integrated our p63 datasets with published bulk and single-cell RNA-sequencing of mouse 4NQO-treated tongue and esophageal tumors, respectively, to generate a p63-driven gene signature that sheds new light on the role of p63 in murine OSCC. Our analyses reveal known and novel players, such as COTL1, that are regulated by p63 and influence various oncogenic processes, including metastasis. The identification of new sets of potential biomarkers and pathways, some of which are functionally conserved in human OSCC and can prognosticate patient survival, offers new avenues for future mechanistic studies.

Head and neck squamous cell carcinoma (HNSCC) is a complex disease of significant morbidity and mortality with poor survival rates that have persisted despite advances in therapy. The lack of improved outcome can be attributed, in part, to a gap in the understanding of the drivers of the disease and the paucity of novel targeted treatments. Although recent studies into the genomic landscapes of HNSCC have highlighted a striking mutational heterogeneity, the identification of pervasive and broadly actionable driver mutations and/or other genetic alterations remains elusive. In parallel with this genetic diversity, the heterogeneity of HNSCC is also manifested in distinct gene expression-defined subtypes. This raises the possibility that these heterogeneous gene expression programs are shaped by regulatory networks that are closely interwoven with an intrinsic and heritable epigenetic state. We thus hypothesize that there exist key oncogenic transcriptional regulators that confer subtype specificity and dependency and that these factors are linked with and potentially configure the epigenetic states of HNSCC. We have examined the distributions of histone modification marks by ChIP-Seq and steady-state transcriptomes by RNA-Seq across several cell lines that represent the subtypes of HNSCC. Interestingly, our molecular profiling demonstrated differential enrichment patterns of enhancer-associated H3K27Ac signals that recapitulate the segregation of cell lines based on subtype-specific differential gene expression profiles. We have further probed the enhancer landscape to identify cis-regulatory networks (CRM) specific to HPV-ve subtypes (basal and mesenchymal) and the HPV+ve (atypical) subtype. Embedded within the regulatory domains of the CRMs, we find the DNA-binding motifs of transcription factor (TF) families, including AP-1, ETS and p53/p63, to be consistently enriched and highly correlated with the expression of specific genes that define a given subtype. To dissect the functional role of enhancers and their regulators in directing expression, we have defined global cistromes of two key HNSCC-enriched TFs, ETS1 and dNp63, by ChIP-seq and RNA-seq experiments in representative cell lines. These studies, boosted by integrated meta-analysis of HNSCC patient datasets, reveal that the master TF-dependent core gene signatures are associated with key cellular processes that are specifically enriched in HNSCC subtypes. This includes EMT and invasion for the mesenchymal, inflammation for the basal, and drug metabolism processes in the HPV+ve atypical subtype. Finally, we have leveraged network analysis and drug discovery algorithms to identify novel subtype-specific small molecules that can potentially disrupt the CRMs in targeted fashion. Our systematic approach to epigenomically define and characterize the hardwired underpinnings of tumor heterogeneity has unveiled TF-dependent pathways and downstream effectors that are primed for future drug discovery and clinical trials. Citation Format: Christian Gluck, Akinsola Oyelakin, Alexandra Glathar, Michael Buck, Rose-Anne Romano, Satrajit Sinha. Genomic and epigenomic analysis reveal a distinct and targetable transcriptional signature of HNSCC subtypes [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr A26.