Alterations In Head And Neck Squamous Cell Carcinoma Research Articles

Inhibition of ATM or ATR in combination with hypo-fractionated radiotherapy leads to a different immunophenotype on transcript and protein level in HNSCC

BackgroundThe treatment of head and neck tumors remains a challenge due to their reduced radiosensitivity. Small molecule kinase inhibitors (smKI) that inhibit the DNA damage response, may increase the radiosensitivity of tumor cells. However, little is known about how the immunophenotype of the tumor cells is modulated thereby. Therefore, we investigated whether the combination of ATM or ATR inhibitors with hypo-fractionated radiotherapy (RT) has a different impact on the expression of immune checkpoint markers (extrinsic), the release of cytokines or the transcriptome (intrinsic) of head and neck squamous cell carcinoma (HNSCC) cells.MethodsThe toxic and immunogenic effects of the smKI AZD0156 (ATMi) and VE-822 (ATRi) in combination with a hypo-fractionated scheme of 2x5Gy RT on HPV-negative (HSC4, Cal-33) and HPV-positive (UM-SCC-47, UD-SCC-2) HNSCC cell lines were analyzed as follows: cell death (necrosis, apoptosis; detected by AnxV/PI), expression of immunostimulatory (ICOS-L, OX40-L, TNFSFR9, CD70) and immunosuppressive (PD-L1, PD-L2, HVEM) checkpoint marker using flow cytometry; the release of cytokines using multiplex ELISA and the gene expression of Cal-33 on mRNA level 48 h post-RT.ResultsCell death was mainly induced by the combination of RT with both inhibitors, but stronger with ATRi. Further, the immune phenotype of cancer cells, not dying from combination therapy itself, is altered predominantly by RT+ATRi in an immune-stimulatory manner by the up-regulation of ICOS-L. However, the analysis of secreted cytokines after treatment of HNSCC cell lines revealed an ambivalent influence of both inhibitors, as we observed the intensified secretion of IL-6 and IL-8 after RT+ATRi. These findings were confirmed by RNAseq analysis and further the stronger immune-suppressive character of RT+ATMi was enlightened. We detected the down-regulation of a central protein of cytoplasmatic sensing pathways of nucleic acids, RIG-1, and found one immune-suppressive target, EDIL3, strongly up-regulated by RT+ATMi.ConclusionIndependent of a restrictive toxicity, the combination of RT + either ATMi or ATRi leads to comprehensive and immune-modulating alterations in HNSCC. This includes pro-inflammatory signaling induced by RT + ATRi but also anti-inflammatory signals. These findings were confirmed by RNAseq analysis, which further highlighted the immune-suppressive nature of RT + ATMi.

The Role of Hedgehog Signaling Pathway in Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is the sixth leading malignancy worldwide, with a poor prognosis and limited treatment options. Molecularly targeted therapies for HNSCC are still lacking. However, recent reports provide novel insights about many molecular alterations in HNSCC that may be useful in future therapies. Therefore, it is necessary to identify new biomarkers that may provide a better prediction of the disease and promising targets for personalized therapy. The poor response of HNSCC to therapy is attributed to a small population of tumor cells called cancer stem cells (CSCs). Growing evidence indicates that the Hedgehog (HH) signaling pathway plays a crucial role in the development and maintenance of head and neck tissues. The HH pathway is normally involved in embryogenesis, stem cell renewal, and tissue regeneration. However, abnormal activation of the HH pathway is also associated with carcinogenesis and CSC regulation. Overactivation of the HH pathway was observed in several tumors, including basal cell carcinoma, that are successfully treated with HH inhibitors. However, clinical studies about HH pathways in HNSCC are still rare. In this review, we summarize the current knowledge and recent advances regarding the HH pathway in HNSCC and discuss its possible implications for prognosis and future therapy.

Loss of Human Leukocyte Antigen and Immune Escape in Head and Neck Cancer.

Cancer cells evade recognition by the immune system to survive. Head and neck squamous cell carcinoma (HNSCC) is characterized by high levels of immune infiltration and mutation-associated neoantigens; therefore, immune evasion is likely to be an important mechanism in HNSCC tumorigenesis and progression. A commonly employed mechanism of immune evasion is downregulation of human leukocyte antigen (HLA) or loss of heterozygosity (LOH) in tumor cells. The objective of this study was to integrate multi-dimensional genomic and transcriptomic data from HNSCC tumors to better understand the clinical and immunologic implications of HLA LOH. Cross-sectional integrated clinical and genomic analysis. Whole-exome sequencing and RNA-sequencing data from 522 tumors profiled in The Cancer Genome Atlas HNSCC cohort were analyzed and integrated with secondary analyses including immune cell deconvolution data. Associations were analyzed with categorical hypothesis testing and multivariable logistic and Cox regression. HLA LOH was a prevalent event that was identified in 53% of HNSCC tumors; in many cases, more than one class I HLA gene was targeted for LOH. HLA LOH was more common in advanced-stage tumors. Tumors with somatic HLA LOH had tumor microenvironments defined by decreased lymphocyte and T cell infiltration. HLA LOH is one of the most prevalent genetic alterations in HNSCC, and is associated with a cold immune microenvironment, suggesting that HLA LOH is a means of immune evasion. It may have value as a predictive biomarker or potential as a cancer cell-specific therapeutic target. 3 Laryngoscope, 134:160-165, 2024.

Abstract 6062: Viral genome structure correlates with patterns of somatic alteration in human papillomavirus associated head and neck squamous cell carcinoma

Abstract Integration of viral DNA into the host genome has been implicated in human papillomavirus (HPV) associated tumorigenesis, and studies in HPV associated head and neck squamous cell carcinoma (HNSCC) attempting to classify viral integration status have reported associations among HPV status, viral load, and overall survival. However, few genomic studies have correlated HPV structure with commonly occurring somatic mutations in HNSCC. Here, we conducted hybrid-capture DNA sequencing using a panel of over 800 cancer related genomic targets, as well as probes specific for the full HPV16 and HPV18 genomes, in a novel cohort of 525 HNSCC cases. Paired reads were aligned to a human reference containing complete viral genomes, including all high-risk HPV types, which allowed for robust identification of HPV(+) tumors and specific HPV type. Variant calling and filtering, followed by pathway analysis of significantly mutated genes, defined the catalog of somatic driver alterations in HNSCC. Viral genome structure, integration status, and viral load were determined via initial copy number analysis of HPV-specific reads, followed by identification of viral/human breakpoints and indels with variant calling algorithms. Manual review was required in some cases due to variability in read coverage and alignment to HPV genome. We identified 252 HPV(+) tumors, the majority of which were HPV16 (n=228, 90%). Analysis of HPV16(+) tumors revealed five classes with variable viral loads: pure episome (EPI, n=54), episomal rearranged (EPI-R, n=51), episomal chimeric (EPI-C, n=22), integrated (INT, n=92), and integrated deleted (INT-A, n=12). Integrated tumors had at least one integration event (INT) and a few exhibited recurrent deletions in the viral genome (INT-A). Episomal samples had either complete HPV genomes (EPI), circular genomes with one or more indels (EPI-R), or circular genomes with HPV and human DNA (EPI-C). Interestingly, PIK3CA, the most frequently mutated gene in HPV(+) HNSCC, was mutated at a higher frequency in all integrated samples compared to all episomal types (44% vs 23%, p=0.005). We also noticed more deleterious mutations in innate immunity genes (HLA-A/B, B2M, TRAF3, BIRC3) in the episomal samples (16% vs 8%), which may allow cells with intact HPV genomes to evade anti-viral immune responses. Additionally, mutations in oxidative stress response genes that lead to the constitutive activation of NRF2 (NFE2L2, CUL3, KEAP1) were more frequent in integrated samples (11% vs 4%), which could confer radiotherapy resistance and a worse prognosis. Overall, using DNA sequencing and integrative genomic analysis in the largest cohort of HPV(+) HNSCC to date, we developed a novel classification method based on viral genome structure which associates with host somatic alterations that may contribute to variable outcomes in HNSCCs. Citation Format: Jeremiah Ray Holt, Xiaobei Zhao, Hyo Young Choi, Paul Little, Angela L. Mazul, Benjamin Wahle, Vonn Walter, Jose P. Zevallos, David Neil Hayes. Viral genome structure correlates with patterns of somatic alteration in human papillomavirus associated head and neck squamous cell carcinoma [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 6062.

Head and Neck Squamous Cell Carcinoma: Risk Factors, Molecular Alterations, Immunology and Peptide Vaccines.

Head and neck squamous cell carcinoma (HNSCC) arises from the epithelial lining of the oral cavity, hypopharynx, oropharynx, and larynx. There are several potential risk factors that cause the generation of HNSCC, including cigarette smoking, alcohol consumption, betel quid chewing, inadequate nutrition, poor oral hygiene, HPV and Epstein–Barr virus, and Candida albicans infections. HNSCC has causative links to both environmental factors and genetic mutations, with the latter playing a more critical role in cancer progression. These molecular changes to epithelial cells include the inactivation of cancer suppressor genes and proto-oncogenes overexpression, resulting in tumour cell proliferation and distant metastasis. HNSCC patients have impaired dendritic cell (DC) and natural killer (NK) cell functions, increased production of higher immune-suppressive molecules, loss of regulatory T cells and co-stimulatory molecules and major histocompatibility complex (MHC) class Ι molecules, lower number of lymphocyte subsets, and a poor response to antigen-presenting cells. At present, the standard treatment modalities for HNSCC patients include surgery, chemotherapy and radiotherapy, and combinatorial therapy. Despite advances in the development of novel treatment modalities over the last few decades, survival rates of HNSCC patients have not increased. To establish effective immunotherapies, a greater understanding of interactions between the immune system and HNSCC is required, and there is a particular need to develop novel therapeutic options. A therapeutic cancer vaccine has been proposed as a promising method to improve outcome by inducing a powerful adaptive immune response that leads to cancer cell elimination. Compared with other vaccines, peptide cancer vaccines are more robust and specific. In the past few years, there have been remarkable achievements in peptide-based vaccines for HNSCC patients. Here, we summarize the latest molecular alterations in HNSCC, explore the immune response to HNSCC, and discuss the latest developments in peptide-based cancer vaccine strategies. This review highlights areas for valuable future research focusing on peptide-based cancer vaccines.

Genomic Alterations in Head and Neck Squamous Cell Carcinoma: Level of Evidence According to ESMO Scale for Clinical Actionability of Molecular Targets (ESCAT)

Development of high-throughput technologies helped to decipher tumor genomic landscapes revealing actionable molecular alterations. We aimed to rank the level of evidence of recurrent actionable molecular alterations in head and neck squamous cell carcinoma (HNSCC) on the basis of the European Society for Medical Oncology (ESMO) Scale for Clinical Actionability of Molecular Targets (ESCAT) to help the clinicians prioritize treatment. We identified actionable alterations in 33 genes. HRAS-activating mutations were ranked in tier IB because of the efficacy of tipifarnib (farnesyltransferase inhibitor) in HRAS-mutated patients with HNSCC (nonrandomized clinical trial). Microsatellite instability (MSI), high tumor mutational burden (TMB), and NTRK fusions were ranked in tier IC because of PD-1 and TRK tyrosine kinase inhibitors tissue-agnostic approvals. CDKN2A-inactivating alterations and EGFR amplification were ranked in tier IIA because of the efficacy of palbociclib (CDK4/6 inhibitor) and afatinib (tyrosine kinase inhibitor) in these respective molecular subgroups in retrospective analyses of clinical trials. Molecular alterations in several genes, including PIK3CA gene, were ranked in tier IIIA because of clinical benefit in other tumor types, whereas molecular alterations in IGF1R and TP53 genes were ranked in tier IVA and tier V, respectively. The most compelling actionable molecular alterations in HNSCC according to ESCAT include HRAS-activating mutations, MSI, high TMB, NTRK fusions, CDKN2A-inactivating alterations, and EGFR amplification.

Signatures of somatic mutations and gene expression from p16INK4A positive head and neck squamous cell carcinomas (HNSCC).

Human papilloma virus (HPV) causes a subset of head and neck squamous cell carcinomas (HNSCC) of the oropharynx. We combined targeted DNA- and genome-wide RNA-sequencing to identify genetic variants and gene expression signatures respectively from patients with HNSCC including oropharyngeal squamous cell carcinomas (OPSCC). DNA and RNA were purified from 35- formalin fixed and paraffin embedded (FFPE) HNSCC tumor samples. Immuno-histochemical evaluation of tumors was performed to determine the expression levels of p16INK4A and classified tumor samples either p16+ or p16-. Using ClearSeq Comprehensive Cancer panel, we examined the distribution of somatic mutations. Somatic single-nucleotide variants (SNV) were called using GATK-Mutect2 ("tumor-only" mode) approach. Using RNA-seq, we identified a catalog of 1,044 and 8 genes as significantly expressed between p16+ and p16-, respectively at FDR 0.05 (5%) and 0.1 (10%). The clinicopathological characteristics of the patients including anatomical site, smoking and survival were analyzed when comparing p16+ and p16- tumors. The majority of tumors (65%) were p16+. Population sequence variant databases, including gnomAD, ExAC, COSMIC and dbSNP, were used to identify the mutational landscape of somatic sequence variants within sequenced genes. Hierarchical clustering of The Cancer Genome Atlas (TCGA) samples based on HPV-status was observed using differentially expressed genes. Using RNA-seq in parallel with targeted DNA-seq, we identified mutational and gene expression signatures characteristic of p16+ and p16- HNSCC. Our gene signatures are consistent with previously published data including TCGA and support the need to further explore the biologic relevance of these alterations in HNSCC.

Comprehensive analysis of the expression and prognosis for CDCAs in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC), a tumor included oral cavity, lips, larynx, oropharynx, and the nasopharynx et al. The cell division cycle-associated (CDCA) protein family (CDCA1-8) critical for normal cell function and cancer cell proliferation. We explored the mutation signatures and expression levels of various CDCAs in detail in HNSCC. A comprehensive bioinformatics analysis pipeline based on copy number and gene expressions data from patients with HNSCC in order to given new insights into the possible functions and distinct prognostics that underlie CDCAs regulation. We compared the transcriptional expression of CDCAs in HNSCC and found significantly elevated mRNA expression of CDCA1-8 in HNSCC tissues across multiple datasets. We also found CDCA5/6/8 are over-expressed both transcriptionally and translationally in patients with HNSCC. Our results suggested that that mRNA levels of CDCA1/2/4/7 related to the prognosis and can be used as a new useful biomarker for predicting the survival of HNSCC patients. The top 5 CDCAs neighboring gene alterations in HNSCCs were found in MYC, STAG1, RAD21, KLHL9 and NDC80. Multivariable Cox proportional hazard model also showed that CD8+ T cells were higher (P<0.05) in HNSCC-HPV-pos patients and that this was related to CDCA1/2/3/4/5/7. This study utilizes online tools to conduct specific gene analyses from free open databases, but our study requires more large-scale genomics research and basic research.

Imaging-Genomic Study of Head and Neck Squamous Cell Carcinoma: Associations Between Radiomic Phenotypes and Genomic Mechanisms via Integration of The Cancer Genome Atlas and The Cancer Imaging Archive.

Recent data suggest that imaging radiomic features of a tumor could be indicative of important genomic biomarkers. Understanding the relationship between radiomic and genomic features is important for basic cancer research and future patient care. We performed a comprehensive study to discover the imaginggenomic associations in head and neck squamous cell carcinoma (HNSCC) and explore the potential of predicting tumor genomic alternations using radiomic features. Our retrospective study integrated whole-genome multiomics data from The Cancer Genome Atlas with matched computed tomography imaging data from The Cancer Imaging Archive for the same set of 126 patients with HNSCC. Linear regression and gene set enrichment analysis were used to identify statistically significant associations between radiomic imaging and genomic features. Random forest classifier was used to predict the status of two key HNSCC molecular biomarkers, human papillomavirus and disruptive TP53 mutation, on the basis of radiomic features. Widespread and statistically significant associations were discovered between genomic features (including microRNA expression, somatic mutations, and transcriptional activity, copy number variations, and promoter region DNA methylation changes of pathways) and radiomic features characterizing the size, shape, and texture of tumor. Prediction of human papillomavirus and TP53 mutation status using radiomic features achieved areas under the receiver operating characteristic curve of 0.71 and 0.641, respectively. Our exploratory study suggests that radiomic features are associated with genomic characteristics at multiple molecular layers in HNSCC and provides justification for continued development of radiomics as biomarkers for relevant genomic alterations in HNSCC.

High P4HA1 expression is an independent prognostic factor for poor overall survival and recurrent-free survival in head and neck squamous cell carcinoma.

BackgroundProlyl 4‐hydroxylase subunit alpha 1 (P4HA1) plays a critical role in modulating the extracellular matrix and promoting tumor progression in various cancers. However, the association between P4HA1 and head and neck squamous cell carcinomas (HNSCC) has not been thoroughly elucidated to date.Methods P4HA1 mRNA and protein expression in cancer and normal tissues were analyzed using The Cancer Genome Atlas (TCGA), Gene Expression Omnibus, and Human Protein Atlas databases. Quantitative PCR was applied to determine P4HA1 mRNA expression levels in 162 paired HNSCC and adjacent normal tissues. The cBioPortal for Cancer Genomics was utilized to explore P4HA1 genetic alterations in HNSCC. Then, KEGG analysis of P4HA1 co‐expressed genes in HNSCC was conducted using ClueGo in Cytoscape.Results P4HA1 mRNA and protein levels were significantly increased in HNSCC tissues compared with normal tissues. High P4HA1 expression in HNSCC tissues was significantly associated with tumor category, lymphatic metastasis and pathological stage. The area under summary receiver operating characteristic curve of TCGA and validation cohort was 0.887 and 0.883, respectively. Moreover, elevated P4HA1 expression was associated with unfavorable OS (HR: 1.728, P = .001) and RFS (HR: 2.025, P = .002) in HNSCC patients.ConclusionsThis integrated analysis provides strong evidence that increasing P4HA1 expression is significantly associated with the carcinogenesis of HNSCC. Additionally, high P4HA1 expression serves as both diagnostic biomarker and independent prognostic factor for poor OS and RFS in HNSCC patients.

Genetic Factors Associated with a Poor Outcome in Head and Neck Cancer Patients Receiving Definitive Chemoradiotherapy.

About half of advanced stage head and neck squamous cell carcinoma (HNSCC) patients can be cured by chemoradiotherapy. Patient outcome may be partially determined by the genetic alterations in HNSCC, rendering these alterations promising candidate prognostic factors and/or therapeutic targets. However, their relevance in patient outcome prognosis remains to be assessed in patients that receive standard-of-care chemoradiotherapy. We therefore tested whether frequent genetic alterations were associated with progression free survival (PFS) in advanced stage HNSCC patients who were uniformly treated with definitive platinum-based chemoradiotherapy. To this end, we performed targeted DNA sequencing on frozen pre-treatment tumor biopsy material from 77 patients with advanced stage oro- and hypopharyngeal carcinoma. This provided somatic point mutation and copy number aberration data of 556 genes. The most frequently mutated genes, TP53 (62%), CCND1 (51%), CDKN2A (30%) and PIK3CA (21%), were not associated with PFS. However, co-occurring CCND1 and CDKN2A mutations were associated with short PFS (HR 2.24, p = 0.028) in HPV-negative tumors. Furthermore, tumor mutational burden (sum of somatic point mutations) showed a trend towards decreased PFS (HR 1.9, p = 0.089), and chromosomal instability (CIN) was associated with shorter PFS (HR 2.3, p = 0.023), independent of HPV status. Our results show that tumor mutational burden, CIN markers, and co-occurring CCND1 and CDKN2A mutations are associated with chemoradiotherapy outcomes in advanced stage oro- and hypopharyngeal HNSCC patients, thereby highlighting their prognostic potential. Given their poor prognosis association and link to biological targets, they may also identify patients for novel targeted therapies and immunotherapies.

Novel genetic alterations and their impact on target therapy response in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is highly variable by tumor site, histologic type, molecular characteristics, and clinical outcome. During recent years, emerging targeted therapies have been focused on driver genes. HNSCC involves several genetic alterations, such as co-occurrence, multiple feedback loops, and cross-talk communications. These different kinds of genetic alterations interact with each other and mediate targeted therapy response. In the current review, it is emphasized that future treatment strategy in HNSCC will not solely be based on “synthetic lethality” approaches directed against overactivated genes. More importantly, biologic, genetic, and epigenetic alterations of HNSCC will be taken into consideration to guide the therapy. The emerging genetic alterations in HNSCC and its effect on targeted therapy response are discussed in detail. Hopefully, novel combination regimens for the treatment of HNSCC can be developed.

Abstract 5884: 4EBP1 is a tumor suppressor gene unleashed by mTOR inhibition in head and neck squamous cell carcinoma

Abstract Persistent aberrant activation of the PI3K/AKT/mTOR signaling pathway occurs in over 80% of head and neck squamous cell carcinomas (HNSCC). In prior studies we have shown that mTOR regulates proliferation, angiogenesis and apoptosis, thus playing an essential role in HNSCC initiation and progression. The frequent over-reliance on this signaling circuit for HNSCC growth may in turn represent a cancer vulnerability that can be exploited therapeutically. Indeed, we have shown that mTOR inhibitors (mTORi) promote tumor regression in multiple genetically defined and chemically induced carcinogenesis animal models, and encouraging results have been recently obtained using mTORi in HNSCC in the clinic. However, the complexity of genomic alterations in HNSCC may facilitate the emergence of drug resistance, especially in cases with patients who may receive multiple rounds of chemotherapy and/or radiation. Understanding the precise molecular processes by which mTOR acts in HNSCC may help predict intrinsic resistance mechanisms and select patients who may benefit the most from the administration of mTORi. Here, we focused on EIF4EBP1 (4EBP1), a direct target of mTOR that serves as key effector for protein synthesis. Remarkably, we found a negative correlation between 4EBP1 gene copy number loss and disease-free survival (p&amp;lt;0.001) and overall survival (p&amp;lt;0.01) in the HNSCC TCGA database (n=181). Furthermore, IHC analysis of 4EBP1 protein expression revealed that 16.3% of HNSCC cases analyzed (n=49) lack 4EBP1, with progressive protein loss in advanced cases. Aligned with a tumor-suppressive role, 4ebp1-knockout mice formed larger and more HNSCC lesions (p&amp;lt;0.001) in 4NQO oral carcinogenesis models. We engineered a mutant 4EBP1 that cannot be phosphorylated by mTOR (4EBP1-mut), resembling de-phospho-4EBP1, and generated HNSCC cells expressing this mutant in a tetracycline-inducible fashion. Protein-protein interactions and assembly of the translational complex were analyzed using 7-methyl GTP pull-down and co-IP assays. Expression of 4EBP1-mut disrupted the function of the translation initiation complex and was sufficient to prevent tumor growth in HNSCC xenograft models. Similarly, mTORi induced the accumulation of dephosphorylated endogenous wild-type 4EBP1 and resulted in HNSCC xenograft regression. Remarkably, CRISPR-CAS9-mediated knockout of 4EBP1 in HNSCC cells caused insensitivity to mTORi in vitro and in vivo. Overall, our findings suggest that 4EBP1 needs to be persistently repressed by mTOR phosphorylation in HNSCC and that mTORi may unleash 4EBP1's tumor-suppressive activity, thereby reducing the translation of growth-promoting and cancer cell stemness-related proteins. In turn, 4EBP1 gene and protein loss may underlie mTORi resistance, supporting that 4EBP1 expression and phosphorylation status may represent a mechanistic biomarker of mTORi sensitivity in HNSCC patients. Citation Format: Zhiyong Wang, Daniel Martin, Lynn Vitale-Cross, Xiaodong Feng, Alfredo A. Molinolo, Michael M. Allevato, Victoria H. Wu, Mara Gilardi, Hao Xu, Qianming Chen, J.Silvio Gutkind. 4EBP1 is a tumor suppressor gene unleashed by mTOR inhibition in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5884.

Abstract IA12: An in vivo high throughput shRNA screening platform for identifying ways to target genomic alterations

Abstract Comprehensive characterization of genomic alterations prevalent in head and neck squamous cell carcinoma (HNSCC) has been performed by our group and others, and more recently by The Cancer Genome Atlas (TCGA) project. A significant barrier towards targeting the genomic alterations collectively found in HNSCC is that the genomic landscape of this disease is dominated by mutations to tumor suppressor genes that are inherently difficult to target, with very few mutated oncogenes present. Although potentially targetable oncogenic drivers may exist within chromosomal regions of copy number gain, the large number of genes within these regions makes identifying the precise targets challenging. To overcome some of these difficulties, we have been leveraging high throughput loss of function shRNA screens performed in vivo in order to identify co-dependencies and potential ways to target genomic alterations in HNSCC. Using a large panel of genomically characterized established HNSCC cell lines in conjunction with several different shRNA libraries, including ones that target druggable genes (i.e. FDAome), genes involved in DNA repair, and candidate HNSCC driver genes, we hope to link shRNA targets that are vital for in vivo growth and survival to specific genomic subtypes present within HNSCC. Following pooled in vitro infection with these libraries, HNSCC cell lines are injected into the flanks of mice where in vivo selection takes place, and dropout of barcoded shRNAs targeting genes advantageous for tumor growth is subsequently analyzed from harvested tumors by next generation sequencing. The FDAome library contains shRNAs to nearly 200 genes immediately targetable by FDA approved drugs, allowing identification of druggable co-dependent pathways through simple lethality. By comparing dropout of shRNAs from tumors of mice treated with or without carboplatin, genes that chemosensitize tumors in vivo are also being identified within the FDAome and DNA repair shRNA libraries. Screens performed with our custom HNSCC shRNAs library targeting many genes from regions recurrently amplified in large subsets of patient tumors should identify which genes within these regions are true drivers and potentially interesting drug candidates. Thus far, we have completed in vivo screens employing the FDAome and DNA damage libraries on four different HNSCC cell lines, comprised of 3 different NOTCH1 mutants and one NOTCH1 wild type cell line, which collectively include two HPV-positive and two HPV-negative cell lines. Data analysis has identified a number of druggable targets that inhibit the growth of NOTCH1 mutant cell lines or that seem to be specific for HPV+ tumors. Some of these targets have been further validated through in vivo experiments employing single shRNAs to genes of interest, employing targeting sequences distinct from the libraries. The growth inhibitory effects for some of the hits are apparent in vivo, but not in vitro, highlighting the ability of this unique platform to identify genes or pathways that drive the three-dimensional growth of tumors in the microenvironment of the host. We have also completed an in vivo screen on two of the cell lines using the custom shRNA library that targets genes recurrently amplified in HNSCC, and are currently analyzing data. Preliminary analysis indicates that this high throughput in vivo shRNA screening platform is a promising and relevant approach for identifying co-dependencies and ways to target genomic alterations in cancer. Citation Format: Tongxin Xie, Ramya L. Parimi, Manish F. Kumar, Liang P. Yang, Jiping Wang, Sahil Seth, Carugo Alessandro, Christopher A. Bristow, Frederick Scott Robinson, Michael D. Peoples, Heath Skinner, Jeffrey N. Myers, Curtis R. Pickering, Mitchell J. Frederick. An in vivo high throughput shRNA screening platform for identifying ways to target genomic alterations [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr IA12.

Abstract B33: The role of WWOX in HNSCC through Wnt/beta-catenin pathway

Abstract The second most common fragile site in the human genome is the FRA16D region where the WWOX gene is localized which codes for a 414 amino acid protein. WWOX is a member of the WW-domain containing protein family consisting of 2000 members. It is a partner of several transcription factors and therefore functions in multiple physiological and pathological processes such as cell growth, differentiation and tumor suppression. Genomic alterations within the WWOX gene and its differential expression have been found in a variety of human tumors. Although some transcription factor partners of the WWOX protein have been identified its exact mechanism of action is not known. A recent study indicated that WWOX inhibits the Wnt/β-catenin pathway by preventing the nuclear import of the Dishvelled (Dvl) proteins. Dvl proteins are the main down-stream target of Wnt receptors (Frizzled- FRZ). Following binding of Wnt to its receptor alterations of β-catenin degradation lead to β-catenin accumulation in the cytoplasm. Accumulated β-catenin translocates into the nucleus and activates expression of target genes. Therefore dysregulation of the Wnt signaling pathway is a key oncogenic mechanism in many different cancers. Although Wnt and its receptor FRZ have been analyzed in head and neck squamous cell carcinoma (HNSCC) the intracellular components of the pathway have not been investigated. This is the first study in the literature reporting a role for β-catenin and Dvl mRNA level alterations in HNSCC. In our previous study we have identified that the WWOX gene is inactivated in HNSCC as a result of genetic and epigenetic alterations. To identify the mechanism of action of WWOX in HNSCC in this study we investigated its expression in association with Dvl-1, Dvl-2, Dvl-3 and β-catenin expression in 98 HNSCC samples. We observed down-regulation of the WWOX mRNA in 73.5% of the HNSCC tumor samples compared to their non-cancerous counterparts. Up-regulation of the Dvl-1, Dvl-2, Dvl-3 and β-catenin mRNAs were detected in 32.7%, 36%, 38.8% and 24.5% of the same tumor samples, respectively. We also detected a statistically significant correlation between the WWOX gene expression and mRNA levels of the intracellular components of the Wnt pathway genes. Our results indicate that WWOX acts as a tumor suppressor in the HNSCC development and progression by inhibiting the expression of the Dvl proteins. Citation Format: Ayse Nur Buyru, Asuman Celebi, Betul Seyhan. The role of WWOX in HNSCC through Wnt/beta-catenin pathway [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr B33.

A 13-gene expression-based radioresistance score highlights the heterogeneity in the response to radiation therapy across HPV-negative HNSCC molecular subtypes

BackgroundRadiotherapy for head and neck squamous cell carcinomas (HNSCC) is associated with a substantial morbidity and inconsistent efficacy. Human papillomavirus (HPV)-positive status is recognized as a marker of increased radiosensitivity. Our goal was to identify molecular markers associated with benefit to radiotherapy in patients with HPV-negative disease.MethodsGene expression profiles from public repositories were downloaded for data mining. Training sets included 421 HPV-negative HNSCC tumors from The Cancer Genome Atlas (TCGA) and 32 HNSCC cell lines with available radiosensitivity data (GSE79368). A radioresistance (RadR) score was computed using the single sample Gene Set Enrichment Analysis tool. The validation sets included two panels of cell lines (NCI-60 and GSE21644) and HPV-negative HNSCC tumor datasets, including 44 (GSE6631), 82 (GSE39366), and 179 (GSE65858) patients, respectively. We finally performed an integrated analysis of the RadR score with known recurrent genomic alterations in HNSCC, patterns of protein expression, biological hallmarks, and patterns of drug sensitivity using TCGA and the E-MTAB-3610 dataset (659 pancancer cell lines, 140 drugs).ResultsWe identified 13 genes differentially expressed between tumor and normal head and neck mucosa that were associated with radioresistance in vitro and in patients. The 13-gene expression-based RadR score was associated with recurrence in patients treated with surgery and adjuvant radiotherapy but not with surgery alone. It was significantly different among different molecular subtypes of HPV-negative HNSCC and was significantly lower in the “atypical” molecular subtype. An integrated analysis of RadR score with genomic alterations, protein expression, biological hallmarks and patterns of drug sensitivity showed a significant association with CCND1 amplification, fibronectin expression, seven hallmarks (including epithelial-to-mesenchymal transition and unfolded protein response), and increased sensitivity to elesclomol, an HSP90 inhibitor.ConclusionsOur study highlights the clinical relevance of the molecular classification of HNSCC and the RadR score to refine radiation strategies in HPV-negative disease.

Abstract 351: mTOR-ERK co-targeting strategies for head and neck cancer therapy

Abstract Despite therapeutic advances in the past decade, head and neck squamous cell carcinoma (HNSCC) continues to be a major public health concern. Although recent studies of human cancer genomes identified remarkable complexity of genomic alterations in HNSCC, surprisingly, most aberrations have fallen within key driver signaling pathways. Among them, PI3K/AKT/mTOR pathway is most frequently activated in over 80% of the HNSCC patients. We showed that the majority of HNSCC lesions exhibit high levels of phosphorylated ribosomal S6 protein (pS6), key downstream target of the AKT-mTOR pathway, and that inhibition of mTOR by the use of rapamycin causes a rapid decrease in the level of pS6 and the apoptotic death of HNSCC tumor xenografts, thereby causing tumor regression. Our initial studies identified the AKT-mTOR pathway as a potential therapeutic target for HNSCC and provided strong rational to initiate multiple clinical trials. Indeed, inhibitors of PI3K/AKT/mTOR block mTOR activity and exert a beneficial response in HNSCC patients. However, therapeutic resistance was evidenced in several trials, which might be due to the activation of adaptive survival signaling in tumor cells. To explore the therapeutic option that can overcome the resistance, we performed a synthetic lethality screen in HNSCC cell lines using shRNA libraries. We found that multiple molecules involved in ERK pathway were highly represented. Furthermore, treating HNSCC cell lines harboring activated mTOR with small molecule inhibitors that target PI3K/AKT/mTOR pathway can sequentially induce ERK activation, which is consistent with the findings observed in numerous clinical trials. We demonstrated that co-targeting mTOR and ERK using trametinib, a MEK1/2 inhibitor or cetuximab, an EGFR monoclonal antibody, exhibited the synergistic effect by sensitizing HNSCC to priming with the mTOR inhibitor. Specifically, mTOR-ERK co-targeting prevented the growth of HNSCC tumor xenografts by decreasing cell proliferation, reducing lymphangiogenesis and activating apoptosis. In addition, we have recently used a phosphoproteomics approach to identify the molecular mechanisms of ERK feedback activation caused by PI3K/AKT/mTOR inhibition. Overall, our promising findings from these preclinical studies suggest that the use of mTOR-ERK co-targeting strategies may provide the novel alternative approach to achieve durable responses (cure) in HNSCC cancer patients. Citation Format: Zhiyong Wang, Esteban Delgado, Kosuke Yamaguchi, Ramiro Iglesias-Bartolomé, Panomwat Amornphimoltham, Alfredo Molinolo, Qianming Chen, J.Silvio Gutkind. mTOR-ERK co-targeting strategies for head and neck cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 351. doi:10.1158/1538-7445.AM2017-351

Abstract 2992: Identification of NOTCH1 inactivating mutation as a therapeutic vulnerability to PI3K/mTOR pathway inhibition in head and neck squamous cell carcinoma (HNSCC)

Abstract Background: Genomic alterations in the PI3K/mTOR pathway occur in 54% of HNSCC patients. However, clinical trials of PI3K/mTOR pathway inhibitors had limited success even in those tumors with pathway alterations, including PIK3CA mutations. To target genomic alterations in HNSCC, we tested the efficacy of 7 PI3K/mTOR pathway inhibitors in 59 HNSCC cell lines and determined the association between drug sensitivity and molecular characteristics in order to identify biomarkers of response. Methods: We systematically analyzed the association between drug sensitivity and genomic alterations in 59 HNSCC lines. Results: NOTCH1mut lines are significantly sensitive to PI3K/mTOR pathway inhibitors: GSK2126458 (13/16), BYL719 (6/16), PQR309 (13/16), BKM120 (14/16), BEZ235 (12/16), BAY806942 (14/16) and GDC0980 (13/16 lines). In contrast to PIK3CAmut cell lines, all 7 NOTCH1mut lines tested underwent apoptosis (14.3 fld; P&amp;lt;0.005). After PI3K/mTOR inhibition, NOTCH1mut lines showed significantly reduced clonogenic growth in vitro (0.4/ 0.9 fold in HN31/ PCI15B; P&amp;lt;0.05) and significant tumor growth inhibition in vivo using orthotopic oral xenograft mouse models (1.7 and 2 fold in UMSCC22A and HN31; P&amp;lt;0.01). To determine if NOTCH1 mediates resistance, we conditionally expressed cleaved NOTCH1 by Dox-inducible system in a NOTCH1mut line (UMSCC22A). This rescued PI3K/mTOR inhibitor-induced apoptosis (0.5 fold; P&amp;lt;0.05) and reduced colony formation in vitro. As no canonical pathways account for the underlying mechanism of sensitivity, we measured the level of 301 proteins by reverse phase protein array (RPPA) in 3 NOTCH1mut and 3 NOTCH1WT lines after GSK2126458 treatment. Glutaminase and Glutamate Dehydrogenase were differentially expressed in NOTCH1mut lines. Thus, we hypothesized that PI3K/mTOR inhibition in NOTCH1mut lines induced reactive oxygen species (ROS)-mediated apoptosis via metabolic alterations. Consistent with this hypothesis, NOTCH1mut lines exhibited increased ROS production; Metabolic pathway inhibitors targeting Glycolysis, Pentose Phosphate pathway and Glutaminolysis, in combination with GSK2126458 decreased cell viability in NOTCHWT lines. Conclusion: In contrast to PIK3CAmut cells, NOTCH1mut HNSCC cells underwent apoptosis after PI3K/mTOR pathway inhibition in vitro and decreased tumor size in vivo. The ectopic activation of NOTCH1 rescued NOTCH1mut HNSCC cells from PI3K/mTOR inhibitor-mediated apoptosis. The underlying mechanism may involve differential effects on tumor metabolism and ROS production. This work is significant because inactivating NOTCH1 mutations, which occur in 18% of HNSCC patients and SCCs of the lung, esophagus, and other sites, may serve as a biomarker for response. Our future work may uncover previously unknown crosstalk between the PI3K/mTOR and NOTCH pathways in SCCs. Citation Format: Vaishnavi Sambandam, Li Shen, Pan Tong, Tuhina Mazumdar, Curtis Pickering, Jeffrey N. Myers, Jing Wang, Mitchell Frederick, Faye M. Johnson. Identification of NOTCH1 inactivating mutation as a therapeutic vulnerability to PI3K/mTOR pathway inhibition in head and neck squamous cell carcinoma (HNSCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2992. doi:10.1158/1538-7445.AM2017-2992

PD-L1: a novel prognostic biomarker in head and neck squamous cell carcinoma.

BackgroundThe PD-1 receptor and its ligands PD-L1 and PD-L2 are known to be significantly involved in T-cell regulation. Recent studies suggest that PD-L1 expression in malignant tumors contributes to an immunosuppressive microenvironment and disruption of antitumoral immune response. Drugs targeting this pathway are already tested in clinical trials against several tumor entities with promising results. However, until now comprehensive data with regard to PD-L1 and PD-L2 expression in head and neck squamous cell carcinoma (HNSCC) is still lacking.Patients and methodsWe assessed PD-L1 and PD-L2 expression via immunohistochemistry in two independent cohorts of 293 HNSCC patients.ResultsA significant subset of HNSCC showed high expression levels of PD-L1. Most remarkable, we detected a strong correlation between PD-L1 expression and overall survival time in both HNSCC cohorts. Further, in multivariate cox proportional hazard models, PD-L1 dominates as the strongest prognostic factor of patient's outcome in HNSCC, leaving even tumor stage and distant metastasis behind. Moreover, strong PD-L1 expression was associated with the presence of distant metastases in a subset of cases.ConclusionsIn summary, while the significance of PD-L2 in HNSCC seems to minor, we show that PD-L1 expression is common in HNSCC and, more importantly, a both robust and strong prognostic biomarker. In this respect, our results provide hints on further application of therapies targeting the PD-1/PD-L1 pathway in HNSCC. Investigation of response and outcome of patients receiving anti-PD-1/PD-L1 containing therapies in correlation with PD-L1 expression analysis should be an important task for the future.STATEMENT OF TRANSLATIONAL RELEVANCEIn spite of improved treatment options and increasing knowledge of molecular alterations in HNSCC, the survival rate has not been dramatically changed in the past decades. Pies are missing in HNSCC. One promising candidate in cancer immune therapy is PD-L1.Drugs targeting PD-L1 or its receptor PD-1 are subject of several clinical studies in different cancer entities. However, comprehensive data about PD-L1 expression in HNSCC and therefore a rational basis for anti PD-L1/PD-1 therapy in HNSCC is lacking. Here, we provide wide-ranging data about PD-L1 expression in HNSCC of all major localizations. We observed a strong correlation between expression of PD-L1 and reduced overall survival time. Furthermore, high PD-L1 expression was identified as a strong prognostic factor of patient's outcome when verified together with recognized prognostic factors.

TP53 Mutations in Head and Neck Squamous Cell Carcinoma and Their Impact on Disease Progression and Treatment Response.

Recent studies describing the mutational landscape of head and neck squamous cell carcinoma (HNSCC) on a genomic scale by our group and others, including The Cancer Genome Atlas, have provided unprecedented perspective for understanding the molecular pathogenesis of HNSCC progression and response to treatment. These studies confirmed that mutations of the TP53 tumor suppressor gene were the most frequent of all somatic genomic alterations in HNSCC, alluding to the importance of the TP53 gene in suppressing the development and progression of this disease. Clinically, TP53 mutations are significantly associated with short survival time and tumor resistance to radiotherapy and chemotherapy in HNSCC patients, which makes the TP53 mutation status a potentially useful molecular factor for risk stratification and predictor of clinical response in these patients. In addition to loss of wild-type p53 function and the dominant-negative effect on the remaining wild-type p53, some p53 mutants often gain oncogenic functions to promote tumorigenesis and progression. Different p53 mutants may possess different gain-of-function properties. Herein, we review the most up-to-date information about TP53 mutations available via The Cancer Genome Atlas-based analysis of HNSCC and discuss our current understanding of the potential tumor-suppressive role of p53, focusing on gain-of-function activities of p53 mutations. We also summarize our knowledge regarding the use of the TP53 mutation status as a potential evaluation or stratification biomarker for prognosis and a predictor of clinical response to radiotherapy and chemotherapy in HNSCC patients. Finally, we discuss possible strategies for targeting HNSCCs bearing TP53 mutations. J. Cell. Biochem. 117: 2682-2692, 2016. © 2016 Wiley Periodicals, Inc.