Head And Neck Squamous Cell Carcinoma Tumors Research Articles

Comparative analysis of the tumor microbiome, molecular profiles, and immune cell abundances by HPV status in mucosal head and neck cancers and their impact on survival

ABSTRACT Head and Neck Squamous Cell Carcinoma (HNSCC) comprises a diverse group of tumors with variable treatment response and prognosis. The tumor microenvironment (TME), which includes microbiome and immune cells, can impact outcomes. Here, we sought to relate the presence of specific microbes, gene expression, and tumor immune infiltration using tumor transcriptomics from The Cancer Genome Atlas (TCGA) and associate these with overall survival (OS). RNA sequencing (RNAseq) from HNSCC tumors in TCGA was processed through the exogenous sequences in tumors and immune cells (exotic) pipeline to identify and quantify low-abundance microbes. The detection of the Papillomaviridae family of viruses assessed HPV status. All statistical analyses were performed using R. A total of 499 RNAseq samples from TCGA were analyzed. HPV was detected in 111 samples (22%), most commonly Alphapapillomavirus 9 (90.1%). The presence of Alphapapillomavirus 9 was associated with improved OS [HR = 0.60 (95%CI: 0.40–0.89, p = .01)]. Among other microbes, Yersinia pseudotuberculosis was associated with the worst survival (HR = 3.88; p = .008), while Pseudomonas viridiflava had the best survival (HR = 0.05; p = .036). Microbial species found more abundant in HPV- tumors included several gram-negative anaerobes. HPV- tumors had a significantly higher abundance of M0 (p < .001) and M2 macrophages (p = .035), while HPV+ tumors had more T regulatory cells (p < .001) and CD8+ T-cells (p < .001). We identified microbes in HNSCC tumor samples significantly associated with survival. A greater abundance of certain anaerobic microbes was seen in HPV tumors and pro-tumorigenic macrophages. These findings suggest that TME can be used to predict patient outcomes and may help identify mechanisms of resistance to systemic therapies.

Aspartate β-Hydroxylase Is Upregulated in Head and Neck Squamous Cell Carcinoma and Regulates Invasiveness in Cancer Cell Models.

Aspartate β-hydroxylase (ASPH) is a protein associated with malignancy in a wide range of tumors. We hypothesize that inhibition of ASPH activity could have anti-tumor properties in patients with head and neck cancer. In this study, we screened tumor tissues of 155 head and neck squamous cell carcinoma (HNSCC) patients for the expression of ASPH using immunohistochemistry. We used an ASPH inhibitor, MO-I-1151, known to inhibit the catalytic activity of ASPH in the endoplasmic reticulum, to show its inhibitory effect on the migration of SCC35 head and neck cancer cells in cell monolayers and in matrix-embedded spheroid co-cultures with primary cancer-associated fibroblast (CAF) CAF 61137 of head and neck origin. We also studied a combined effect of MO-I-1151 and HfFucCS, an inhibitor of invasion-blocking heparan 6-O-endosulfatase activity. We found ASPH was upregulated in HNSCC tumors compared to the adjacent normal tissues. ASPH was uniformly high in expression, irrespective of tumor stage. High expression of ASPH in tumors led us to consider it as a therapeutic target in cell line models. ASPH inhibitor MO-I-1151 had significant effects on reducing migration and invasion of head and neck cancer cells, both in monolayers and matrix-embedded spheroids. The combination of the two enzyme inhibitors showed an additive effect on restricting invasion in the HNSCC cell monolayers and in the CAF-containing co-culture spheroids. We identify ASPH as an abundant protein in HNSCC tumors. Targeting ASPH with inhibitor MO-I-1151 effectively reduces CAF-mediated cellular invasion in cancer cell models. We propose that the additive effect of MO-I-1151 with HfFucCS, an inhibitor of heparan 6-O-endosulfatases, on HNSCC cells could improve interventions and needs to be further explored.

Therapeutic Targeting of Thioredoxin Reductase 1 Causes Ferroptosis while Potentiating Anti-PD-1 Efficacy in Head and Neck Cancer

Head and neck squamous cell carcinoma (HNSCC) faces low response rates to anti-PD-1 immunotherapies, highlighting the need for enhanced treatment strategies. Auranofin, which inhibits thioredoxin reductase (TrxR) through its gold-based composition, has shown potential in cancer treatment. It targets the TrxR system, essential for safeguarding cells from oxidative stress. The overproduction of TrxR in cancerous cells supports their proliferation. However, auranofin's interference with this system can upset the cellular redox equilibrium, boost levels of reactive oxygen species, and trigger the death of cancer cells. This study is the first to highlight TXNRD1 as a crucial factor contributing to resistance to anti-PD-1 treatment in HNSCC. In this study, we identified targetable regulators of resistance to immunotherapy-induced ferroptosis in HNSCC. We observed a link of thioredoxin reductase 1 (TXNRD1) with tumoral PD-L1 expression and ferroptosis suppression in HNSCC. Moreover, HNSCC tumors with aberrant TXNRD1 expression exhibited a lack of PD-1 response, NRF2 overexpression, and PD-L1 upregulation. TXNRD1 inhibition promoted ferroptosis in HNSCC cells with NRF2 activation and in organoid tumors derived from patients lacking a PD-1 response. Mechanistically, TXNRD1 regulated PD-L1 transcription and maintained the redox balance by binding to ribonucleotide reductase regulatory subunit M2 (RRM2). TXNRD1 expression disruption sensitized HNSCC cells to anti-PD-1–mediated Jurkat T-cell activation, promoting tumor killing through ferroptosis. Moreover, TXNRD1 inhibition through auranofin cotreatment synergized with anti-PD-1 therapy to potentiate immunotherapy-mediated ferroptosis by mediating CD8+ T-cell infiltration and downregulating PD-L1 expression. Our findings indicate that targeting TXNRD1 is a promising therapeutic strategy for improving immunotherapy outcomes in patients with HNSCC.

Optimizing Treatment Quality in Head and Neck Cancer Therapy: Impact of Adaptive Intensity-Modulated Radiotherapy on Plan Quality and Tumor Volume Changes

Objective: This research analyses whether volumetric changes and PTV shifting affect adaptive intensity-modulated radiotherapy (IMRT) plan quality and head and neck cancer therapy. Methods: The research was carried out at the Sohag Cancer Centre in Egypt, including a sample of 36 individuals diagnosed with head and neck squamous cell carcinoma (HNSCC). Patients were chosen on the basis of the following criteria: locally progressed and node-positive malignancy, oropharyngeal cancer, oral cavity cancer, and unknown primary. The research used dynamic intensity-modulated radiation therapy (D-IMRT) to establish treatment plans and Siemens SOMATOM DEFINITION CT scanners to acquire 3D anatomical images. A range of 1.64 to 2.12 Gy was observed for the median dosage per fraction. Using the conformance index (CI) and the homogeneity index, the quality of the plan was assessed (HI). The threshold for statistical significance was set at 0.05. Results: The study analysed patients with unilateral and bilateral HNSCC tumors, with a majority having bilateral tumors. The findings demonstrated that the tumor PTV was significantly reduced as a consequence of the suggested adaptive radiation techniques. Both the homogeneity index and the conformity index were used to evaluate the plan's quality (HI). The maximal homogeneity index (HI) was reached after 7 and 14 fractions, respectively. The positive correlation between PTV reduction and CI after 7 fractions, while a negative correlation found after fractions 14 and 21. It was also shown that HI is positively correlated with PTV changes after 7 and 14. Conclusion: The use of adaptive radiotherapy in intensity-modulated radiation therapy (IMRT) treatment planning improves plan quality and decreases error rates due to tumor margin movement. Following radiation fractions, PTV shifting affects the homogeneity index (HI).

Th2 Cells Are Associated with Tumor Recurrence Following Radiation.

The curative treatment of multiple solid tumors, including head and neck squamous cell carcinoma (HNSCC), utilizes radiation. The outcomes for HPV/p16-negative HNSCC are significantly worse than HPV/p16-positive tumors, with increased radiation resistance leading to worse locoregional recurrence (LRR) and ultimately death. This study analyzed the relationship between immune function and outcomes following radiation in HPV/p16-negative tumors to identify mechanisms of radiation resistance and prognostic immune biomarkers. A discovery cohort of 94 patients with HNSCC treated uniformly with surgery and adjuvant radiation and a validation cohort of 97 similarly treated patients were utilized. Tumor immune infiltrates were derived from RNAseq gene expression. The immune cell types significantly associated with outcomes in the discovery cohort were examined in the independent validation cohort. A positive association between high Th2 infiltration and LRR was identified in the discovery cohort and validated in the validation cohort. Tumor mutations in CREBBP/EP300 and CASP8 were significantly associated with Th2 infiltration. A pathway analysis of genes correlated with Th2 cells revealed the potential repression of the antitumor immune response and the activation of BRCA1-associated DNA damage repair in multiple cohorts. The Th2 infiltrates were enriched in the HPV/p16-negative HNSCC tumors and associated with LRR and mutations in CASP8, CREBBP/EP300, and pathways previously shown to impact the response to radiation.

Elucidating the role of FOS in modulating the immune microenvironment through fibroblast and myeloid cell regulation in locoregional recurrent HNSCC.

Head and neck squamous cell carcinoma (HNSCC) presents a significant clinical challenge, particularly due to its high propensity for locoregional recurrence. Current research underscores the need to unravel the complex interactions within the tumor microenvironment. This study addresses the critical gap in understanding how FOS modulates the immune landscape in HNSCC, with a focus on its influence on fibroblast and myeloid cell dynamics. Employing a comprehensive approach, we analyzed tissue samples from HNSCC patients and adjacent non-cancerous tissues using bulk RNA sequencing complemented by in-depth bioinformatics analyses, including gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis, and immune infiltration assessment. A pivotal aspect of our research involved dissecting single-cell RNA-seq data from GSE234933 to elucidate the cell-type-specific expression of FOS. We found that FOS expression varies significantly in different cell populations in the HNSCC tumor microenvironment, especially in fibroblasts and myeloid cells. This expression difference may reflect the different roles of these cells in tumor progression and their impact on the tumor microenvironment. Our results uncover a significant correlation between FOS expression and key immune and hypoxia-related pathways, suggesting its integral role in the tumor microenvironment. These findings not only enhance our understanding of HNSCC pathogenesis but also highlight FOS as a potential therapeutic target. This study marks a significant step towards addressing the urgent need for targeted interventions in HNSCC, particularly in the context of locoregional recurrence.

Abstract 4352: A high rate of episomal HPV16 is present in head and neck squamous cell carcinoma tumors by long-read whole genome sequencing

Abstract Persistent Human Papillomavirus (HPV) infection causes an increased risk of Head and Neck Squamous Cell Carcinoma (HNSCC). However, a comprehensive understanding of the intricate molecular events within virus-positive tumors remains a critical knowledge gap. Therefore, we used long-read sequencing to study the intricacies of HPV integration events within the genomic landscape of HPV+ HNSCC tumors. A total of 16 tissue biopsies, including 10 tumors and 6 matched adjacent normal tissues from histopathologically confirmed HNSCC subjects were sequenced by LSK114 chemistry of Oxford Nanopore Technology with an average coverage of 30X and 10X, respectively. The predicted mitochondrial haplotype from Mitomaster confirmed that the tumor/normal pair was from the same individual. The HPV integration status was determined by aligning fastq_pass files at the Cancer Genomics Cloud and viewing the breakpoints in the Integrated Genome Viewer. HPV lineage and sub-lineage classification was determined using ClustalOmega. We detected HPV16 in 8/10 tumors, with sublineages A1 and A2 accounting for 38% (3/8) and D3 for 25% (2/8). One tumor sample had a very low HPV load with only one HPV16 sequence read and low detection by PCR. Interestingly, we found that 5/8 HPV positive tumors had episomal HPV16 with different states. So, two tumors had intact HPV episomes, two more had either a small deletion or an insertion in the episomal HPV, and the last one had a rearranged episomal multimer (Table 1). The remaining three tumors had integrated HPV16: one with type 1 integrations at 11q12.13 and two others with type 2 integrations at chr1q42.12 and chr14q21.1. Our results indicate that episomal HPV16 is more prevalent than integrated HPV16 in primary HPV+ HNSCC samples. These findings provide new insights into the molecular mechanisms and consequences of HPV infection in HNSCC. Table 1: HPV characterization in HNSCC studied tissue biopsies. Patient ID Race HPV status HPV sublineages Predicted Haplotype HPV pattern #PID_1 Caucasian HPV16 + A2 T2b (T2b) Type 2 Integration (chr1q42.12) #PID_2 - HPV- - I1a (I1a1b) - #PID_3 Caucasian HPV16 + A1 H1q (H1q) Episomal (rearranged multimer) PID_4 African American HPV16 + A1 - Episomal (deletion of 636bp from 4892 to 5528) #PID_5 - HPV16 + A2 H1 (H1+16189) Type 1 Integration (complex chr11q12.13) *#PID_6 Caucasian HPV16 + - T2b (T2b) - PID_7 Caucasian HPV16 + D3 - Episomal #PID_8 African American HPV16 + D3 L1c (L1c3a) Type 2 Integration (chr14q21.1) PID_9 African American HPV16 + A2 - Episomal (insertion of 257bp from 7310-7567) PID_10 Caucasian HPV16 + A1 - Episomal *Very weakly HPV16 positive; #Tumor-normal pair Citation Format: Sonam Tulsyan, Vera Mukhina, Isabel Rodriguez, Subhendu R. Choudhury, Erin Allor, Kyle Hatte, Michael Dean, Daria A. Gaykalova. A high rate of episomal HPV16 is present in head and neck squamous cell carcinoma tumors by long-read whole genome sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4352.

Abstract 2466: tRNA-derived fragments as immunotherapy response targets in head and neck squamous cell carcinoma

Abstract Background: Despite significant advances in immunotherapy-based treatment options for Head and Neck Squamous Cell Carcinoma (HNSCC), patient prognosis and survival remains poor. To enhance response to immune-based therapies, specific targeting of immunomodulatory pathways has been investigated through co-stimulatory gene targets, the microbiome, and more. An emerging biomolecule is Transfer RNAs (tRNAs), which are a class of RNAs that transport amino acids from the cytoplasm to ribosomes, and synthesize new proteins encoded by mRNA transcripts. tRNA-derived fragments (tRFs) are a category of cleaved, mature tRNA that participate in post-transcriptional regulation and have been implicated in the modulation of cancer and other immune-associated diseases. While tRFs are primarily implicated in diagnostic signatures for various cancers, few studies have investigated their role in modulating the immunotherapy-based response in HNSCC. In this study, we characterized the role of tRFs in modulating immunotherapy gene targets pertinent to HNSCC. Methods: We extracted tRF read counts from MINTbase 2.0 for 455 primary HNSCC tumor samples and 44 adjacent normals. Differential expression analysis was first performed to identify tRFs implicated in HNSCC tumors, with respect to adjacent normal tissue. Next, these tRFs were associated with immunotherapy target genes and response markers with Spearman’s correlation. Further characterization of these tRFs was performed by correlating their abundance to infiltrating immune cells, pathway enrichment of immunotherapy targets, and clinical variables. Results: Significant differential expression of tRFs were found between HNSCC primary tumor samples and adjacent normal tissue, including 7 tRFs (FDR &amp;lt; 0.05, |log fold change| &amp;gt; 0.5). All were enriched in normal samples. AlaCGC5tRF appeared to be an important regulator of immunotherapy target genes through increased co-expression of PTPN11, STAT3, and PTEN (p-value &amp;lt; 0.05, R &amp;gt; 0) and decreased co-expression of CD58 and TNFRSF14 (p-value &amp;lt; 0.05, R &amp;lt; 0). Immune Checkpoint Inhibitor (ICI) genes were significantly correlated with several tRFs. Among several pertinent tRF markers, AlaCGC5tRF was strongly associated with negatively correlated with infiltrating activated NK cells naïve CD4 T cells (p-value &amp;lt; 0.05, R &amp;lt; 0). Conclusion: Our study is the most comprehensive to date to characterize the role of tRF elements in modulating HNSCC immunotherapy targets. We found several potential tRF biomarkers that may be indicative of enhanced immunotherapy treatment susceptibility, including AlaCGC5tRF and ArgTCT3tRf. Further studies are needed to investigate how these tRFs may be targeted alongside immunotherapies to enhance response to treatment. Citation Format: Rishabh Yalamarty, Daniel John, Annie Do, Tianyi Chen, Megan Ngo, Pranava Gande, Matthew Uzelac, Wei Tse Li, Weg M. Ongkeko. tRNA-derived fragments as immunotherapy response targets in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2466.

Abstract 6742: PV 10 induces endoplasmic reticulum stress and autophagy, triggering immunogenic cell death and anti-tumor immunity in head and neck squamous cell carcinoma

Abstract Introduction: Head and neck squamous cell carcinoma (HNSCC) are complex, diverse cancers affecting mucosal linings of the upper aerodigestive tract. Globally, HNSCC is the sixth most common cancer, leading to over 890,000 new cases and more than 450,000 deaths annually. Primary risk factors include human papillomavirus (HPV) infection (HPV-positive HNSCC) and exposure to tobacco and excessive alcohol (HPV-negative HNSCC). Despite multimodal treatments, survival rates remain low due to recurrence and metastasis (R/M HNSCC), creating a need for safe, innovative therapies to reduce tumor burden with minimal adverse events. PV-10 (10% rose bengal sodium) is a novel intratumoral immunotherapeutic agent that has demonstrated promising anti-tumor activity in multiple solid tumor cancer types with few adverse effects in pre-clinical research and clinical trials. However, the role of PV-10 in HNSCCs and its role in enhancing immune response warrants further investigation. Methods: Cell viability was assessed using Alamar blue assay after PV-10 treatment of murine mEER and MTE-RAS HNSCC tumor cell lines. To evaluate the immunomodulatory properties of PV-10, we employed flow cytometry, confocal analysis, and luminescence-based assays. Additionally, we performed a DCFDA assay to measure the reactive oxygen species (ROS) and an Alexa Fluor 488 Annexin V/dead cell apoptosis assay to evaluate apoptosis. To gain insights into the mechanisms underlying PV-10's immunomodulatory effects in cancer cells and tumors from mice, western blotting and multiplex immunohistochemistry (mIHC) were used. Results: Our in vitro findings reveal that PV-10 induces cytotoxicity in both mEER and MTE-RAS cells. Notably, PV-10 promotes a significant increase in ROS, leading to an elevation in late apoptotic cells. Markers of immunogenic cell death (ICD), including a statistically significant increase in the release of damage-associated molecular pattern molecules HMGB1 and ATP, as well as enhanced surface expression of calreticulin, HSP-70, and HSP-90, were observed. At the molecular level, a remarkable activation of endoplasmic reticulum (ER) stress, pro-apoptotic protein, and autophagy markers were observed. Intratumoral PV-10 injection in vivo has shown significant tumor regression in both mEER and MTE-RAS tumors, and a complete response was noticed in some mice, indicating that PV-10 induces potent ICD in both mEER and MTE-RAS tumors. Conclusion: Our collated evidence strongly suggests that PV-10 induces ICD-associated immunogenicity in both mEER and MTE-RAS in vitro and in vivo, fostered by ROS-based ER stress and apoptosis. These findings hold promise for potential therapeutic avenues to manage HNSCC. Citation Format: Sowjanya Thatikonda, Ritu Chaudhary, Yeva Meshkovska, Maria I. Biernacki, Robbert Slebos, Shari P. Thomas, Amelio L. Antonio, Eric A. Wachter, Christine H. Chung. PV 10 induces endoplasmic reticulum stress and autophagy, triggering immunogenic cell death and anti-tumor immunity in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6742.

Abstract 224: Combining functional precision medicine and radiotherapy with bio-printed organoids in head and neck squamous cell carcinoma

Abstract The study aims to establish a high throughput ex vivo organoid-based drug screening platform for head and neck squamous cell carcinoma (HNSCC) to investigate interactions between radiotherapy and different chemotherapies or targeted therapy agents and ultimately provide functional precision medicine therapeutic suggestions. HNSCC is the sixth most common cancer worldwide. Both HPV+ and HPV- cancers can be treated with surgery, radiotherapy, chemotherapy, or a combination of these. Cetuximab is currently the only FDA-approved targeted agent for HNSCC. Few clinical trials to date have shown an improved clinical outcome when combining targeted agents with radiotherapy, thus there is a need to identify new therapeutic combinations to improve outcomes. In order to characterize HNSCC tumor behavior and sensitivity to therapy, we have leveraged our high throughput 3D tumor organoid screening platform. Our approach entails seeding tumor cells in extracellular matrix around the perimeter of tissue culture wells in ring-like structures, which we have successfully deployed in other cancer types (Phan et al, 2019; Al Shihabi et al, 2022; Nguyen et al, 2022; Al Shihabi et al 2023). To optimize a chemoradiation platform, we used the HN30, HN31, and SCC154 cell lines representing HPV- primary, HPV- metastatic, and HPV+ head and neck tumors. The cell-line-derived 3D models are exposed to both radiotherapy and various chemotherapy or targeted therapy drugs. We measured cell responses by ATP-release assays and by applying our machine learning-based brightfield image analysis pipeline (Al Shihabi et al, 2022). Our results showed that the HPV+ SCC154 was the most sensitive line to radiotherapy while HPV- metastatic HN31 was the most resistant. HN31 was resistant to most drug treatments except for gemcitabine, docetaxel, and mitomycin c, while HN30 and SCC154 were sensitive to a broader set of agents. We have investigated combinatorial efficacy of radiation and chemotherapy as well as targeted agents and extended this platform to HNSCC patient-derived tumor organoids. Our proof-of-concept study highlights how our organoid-based drug screening platform can be used to test chemoradiation approaches that are more effective and personalized. Citation Format: Luda Lin, Krishna K. Bommakanti, Andreanne V. Sannajust, Yazeed Alhiyari, Brandon Mo, Lauran Evans, Peyton Tebon, Maie St. John, Alice Soragni. Combining functional precision medicine and radiotherapy with bio-printed organoids in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 224.

Abstract 2636: Epigenetic landscape of CD8+ T cell exhaustion and tissue residency in HPV-negative head and neck squamous cell carcinoma

Abstract Background Head and neck cancer is the 8th most common cancer worldwide. Human Papilloma Virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) overall survival rates remain low (25-40%) despite advances in treatments. CD8+ T cell differentiation into activated/cytotoxic (CTL), exhausted (TEX) and tissue-resident memory (TRM) phenotypes is a major determinant of successful cancer immunotherapy, which relies on the ability of CD8+ T cells to detect and eliminate cancer cells. Recent evidence suggests the importance of epigenetic regulation in the development of CD8+ T cell exhaustion and tissue residency; yet the epigenetic mechanisms that govern the transition between CTL, TEX, and TRM CD8+ T cell states remain unclear. This study investigates the epigenetic modifications associated with CTL, TEX, and TRM states using CD8+ T cells from healthy donors and HPV-negative HNSCC tumors. Methods An in vitro human CD8+ T cell stimulation assay using healthy donor cells was utilized to generate CTL, TEX and TRM phenotypes. Naïve T cells underwent CD3/28, TCR-independent stimulation for 9 days to promote CTL (3 days) and TEX (9 days) phenotypes, and TGF-β exposure (days 3-9) occurred to induce a TRM phenotype. Flow cytometry to confirm expression of cell-surface and intracellular markers for each phenotype is ongoing. Genome-wide mapping for activating (H3K4me1, H3K4me3) and repressive (H3K9me3, H3K27me3, H4K20me3) histone marks with CUT&amp;Tag assays will be pursued for each of the in vitro CTL, TEX and TRM states, along with RNA-seq. CUT&amp;Tag and RNA-seq will also characterize the epigenetic profiles of CD8+ T cells from single-cell suspensions generated from HPV-negative HNSCC tumors (n=5). Findings will be correlated with results from our in vitro T cell states. Results Preliminary flow cytometry confirmed increased expression of CD25, PD-1, Tim3 after 9 days of in vitro stimulation, supporting the induction of a CD8+ TEX phenotype. CD103 was also highly expressed in CD8+ T cells exposed to TGF-β, supporting the induction of the CD8+ TRM phenotype. Validation of the CTL state is ongoing. Phenotypic validation of cell states with flow cytometry (18 cell surface and intracellular markers) is ongoing. Feasibility of genome-wide mapping for activating and repressive histone marks with CUT&amp;Tag on naïve CD8+ T cells has been confirmed. Conclusion This study investigates epigenetic changes that characterize the transition between CTL, TEX, and TRM states of CD8+ T cells using healthy donor cells through an in vitro assay and CD8+ T cells isolated from HPV-negative HNSCC tumors. Understanding the epigenetic profile of T cell states can help determine the major epigenetic regulators that lead to exhaustion and tissue residency, paving the way for improved T cell-based immunotherapies. Future experiments aim to validate our findings using TCR-specific in vitro and in vivo models. Citation Format: Marie K. Luff, Marco Craviero, Mohd Saleem Dar, Katherine McKinnon, Amir Kaskas, Clint T. Allen, Vassiliki Saloura. Epigenetic landscape of CD8+ T cell exhaustion and tissue residency in HPV-negative head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2636.

Abstract 4335: High-resolution characterization of age-specific cell type composition changes and signaling events in HPV-negative HNSCC tumors

Abstract This study is aimed at testing the hypothesis that increased patient age fosters conditions that are favorable for head and neck squamous cell carcinomas (HNSCC) of the oral cavity development. There is a well-documented increase in cancer incidence with age observed across diverse cancer types, where elderly patients exhibit diminished survival outcomes compared to their younger counterparts. While the convergence of aging and cancer hallmarks offers valuable insights, further work is needed to elucidate the age-specific mechanisms influencing HNSCC, beyond the shared characteristics of these biological processes. To this end, we have assembled a high-quality human single-cell RNA-sequencing HNSCC atlas profiling more than 230,000 cells across more than 50 patients with ages ranging between 18 and 89, which provides a resource to investigate age-associated changes in the disease heterogeneity. For this library, we integrated six publicly available single-cell RNAseq datasets from 54 HPV-negative patients to create the atlas. Cells were clustered, classified, characterized by gene set enrichment analysis, both in the epithelial cell compartment and in the tumor microenvironment (TME). Differential cell type proportion analysis was performed to identify cell types enriched in young (&amp;lt;49 years) or old (&amp;gt;70 years) patients. Cell-cell communication analysis was performed to identify signaling events occurring between different populations, and how these and specific ligand-receptor pairs differed in patients of different ages. CNV analyses were performed to identify cancer subclones and assess level of variation across tumors. Interestingly, we identified distinct cell populations and signaling events that associate with age, and we were able to compare, validate and recapitulate these observations in the 4MOSC1 OSCC isograft tumor model in old and young mice. Additional notable findings include a differential enrichment for cytotoxic CD8 T cells over dysfunctional CD8 T cells in young patients when compared to old, suggesting younger patients have a more effective immune TME. We also found an enrichment for cancer-associated fibroblast populations in old patients (myCAFs) that send collagen signals to malignant epithelial cells, a result we have recapitulated using the 4MOSC1 model, and hypothesize may play a role in ECM stiffening, EMT, and metastasis. Further analyses are ongoing, and we plan to validate the hypotheses generated, specifically the presence of differential abundance of specific cell populations, and age-specific ligand-receptor signaling events that lead to tumor growth. Citation Format: Lina Kroehling, Anthony Spinella, Maria Kukuruzinska, Xaralabos Varelas, Stefano Monti. High-resolution characterization of age-specific cell type composition changes and signaling events in HPV-negative HNSCC tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4335.

Abstract 6658: TcE armed oncolytic virus VSV-GP combines oncolysis and T-cell retargeting for improved efficacy

Abstract Background: Oncolytic viruses, such as the chimeric vesicular stomatitis virus variant VSV-GP have dual modes of action, direct tumor-cell killing, and activation of tumor-specific immunity. While tumor cells deficient in type I interferon (IFN) response are permissive to VSV-GP replication and killing, IFN competent tumor cells are protected from VSV-GP-mediated oncolysis. Our previous data demonstrated that VSV-GP therapy turns immunologically ‘cold’ tumors ‘hot’ by increasing infiltration of T cells into the tumor. The aim of this study was to use the VSV-GP platform to locally express a T cell engager (TcE) molecule to redirect these T cells to destroy non-permissive IFN competent tumor cells expressing the target of the TcE on their cell surface. Methods: We selected EpCAM as the target antigen and used a bispecific EpCAM-TcE molecule consisting of two variable single chain fragments against EpCAM and CD3, respectively. VSV-GP encoding EpCAM-TcE (VSV-GP-EpCAM-TcE) or an invariant control TcE (VSV-GP-TNP-TcE) were generated as previously described. T cells were co-cultured with human target cells expressing EpCAM on their cell surface, either infected with the VSV-GP-EpCAM-TcE virus or with supernatant from VSV-GP-EpCAM-TcE infected cells. T cell activation, proliferation, degranulation and cytokine secretion were analyzed by flow cytometry and multiplex cytokine assays as a measure of TcE mediated cross-linking of T cells to their target on cancer cells. Patient-derived head and neck squamous cell carcinoma (HNSCC) tumors were sectioned into approximately 200 µM slices and infected with the virus. T cell activation was measured by cytokine secretion. The therapeutic potential of VSV-GP-EpCAM-TcE was tested in vivo in cell-line derived xenograft (CDX) models implanted in immunodeficient mice humanized with donor-derived PBMCs or CD34+ cells. Results: Virus fitness was maintained for VSV-GP encoding the TcE molecule. Using co-culture assays, we demonstrated that EpCAM-TcE molecule is successfully expressed by VSV-GP and can activate both CD4+ and CD8+ T cells resulting in proliferation, degranulation, and cytokine secretion. Furthermore, we validated the concept using patient-derived HNSCC tumor slices which are representative of complex human tumor ecosystems. We could confirm that cancer cells infected by VSV-GP-EpCAM-TcE virus express functional TcE molecule and activates T cells to secrete IFNγ and TNFα. Finally, we tested the concept in two different CDX models where VSV-GP-EpCAM-TcE showed improved tumor growth inhibition due to a combined effect of oncolysis and T cell activation by EpCAM-CD3 TcE. Summary: The oncolytic virus VSV-GP can be successfully armed with a TcE to redirect tumor-infiltrating T cells to destroy tumor cells which are not permissive to viral replication. This resulted in an enhanced therapeutic efficacy compared to just the oncolytic effect. Citation Format: Aparna Ponnurangam, Theresa Schwaiger, Sophie Walter, Simone Bartl, Melissa Mayr, Monika Petersson, Tanja Schoenberger, Fabian Heinemann, Peter Gross, Wolfgang Rist, Srinath Kasturirangan, Charlotte Lempp, Jutta Petschenka, Krishna Das, Nolden Tobias. TcE armed oncolytic virus VSV-GP combines oncolysis and T-cell retargeting for improved efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6658.

Abstract 4206: Regional transcriptomic profiling of the head and neck squamous cell carcinoma tumor microenvironment paired with murine models identify extracellular matrix components that mediate tumor progression and immune suppression

Abstract Purpose: A substantial contribution to the poor prognosis for head and neck squamous cell carcinoma (HNSCC) patients is the progression to distant metastasis. This occurs in 30% of patients over the course of their disease, and radically reduces the 5-year survival rate to under 50%. Investigations in recent years have begun to unveil the intricate interplay of the extracellular matrix (ECM) in both tumor-intrinsic signaling and immunoregulation. However, studies conducted in the context of metastatic have been limited. Here, we employ spatial transcriptomics on HNSCC patient biopsies to identify ECM components that may contribute to cancer progression and immune suppression. Experimental design: Comprehensive transcriptome profiling of HNSCC patient biopsies was conducted using the GeoMx Digital Spatial Profiler (DSP). This approach allowed us to characterize regions of the tumor microenvironment (TME) by immunofluorescent staining for subsequent bioinformatic analysis. To investigate the biological consequences of associations observed in our dataset, we performed in vivo murine studies. We have produced a series of SCC C57BL/6 murine cell lines derived from Smad4-/-KrasG12D Keratin15+ stem cells, which faithfully mimic the clinical progression of HNSCC tumors, including metastasis. Using these murine models, we applied relevant pharmacologic inhibitors and genetic knockdown of targets identified with our GeoMx DSP analysis. In vitro immune-based and functional assays were conducted to elucidate the underlying mechanisms contributing to SCC progression. Results: GeoMx DSP analysis revealed that laminins were enriched in stage IV HNSCC patient stroma, relative to lower stage patients. This coincided with TGFβ enrichment and inversely associated with signatures of anti-tumor immune infiltration. Analysis of The Cancer Genome Atlas data revealed similar inverse correlations in SCCs, distinct from breast cancer and lung adenocarcinoma. Our metastatic SCC murine lines likewise exhibited elevated levels of these laminin proteins, accompanied by high levels of laminin-binding integrins and TGFβ-Smad signaling. Independent of impacting cell proliferation, shRNA knockdown of laminin decreased tumor volume in an immune-competent setting. Targeting laminin-integrin signaling with the small molecule inhibitors Galunisertnib and Buparlisib also reduced the metastatic capacity of these cells in C57BL/6 mice. Conclusions: Spatial transcriptomic profiling of the HNSCC TME uncovers intricate associations and promising targets not readily discernible in bulk RNAseq analyses. Validation through genetic and pharmacological approaches in immune-competent models underscores their potential as strategies to reduce HNSCC burden. Citation Format: John D. Aleman, Khoa A. Nguyen, Erica Wong, Yao Ke, Hanne T. Lind, Clifford G. Tepper, Andrew C. Birkeland, Christian D. Young, Sana D. Karam, Xiao-Jing Wang. Regional transcriptomic profiling of the head and neck squamous cell carcinoma tumor microenvironment paired with murine models identify extracellular matrix components that mediate tumor progression and immune suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4206.

Abstract 1729: SUV420H1 depletion reveals therapeutic potential in HPV-negative head and neck squamous cell carcinoma

Abstract Human Papilloma Virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) poses a significant clinical challenge with limited treatment options. SUV420H1, which encodes for a protein lysine methyltransferase (PMT) that methylates H4K20 (H4K20me3), is recurrently amplified in ~5% of HPV-negative HNSCC tumors and is the second most frequently amplified PMT in this disease, suggesting an oncogenic function in this subset of HPV-negative HNSCC patients. Gene set enrichment analysis using the TCGA database of HPV-negative HNSCC tumors revealed that SUV420H1 overexpressing tumors showed enrichment in the mitotic spindle related pathways, and repression of immune-related pathways. To assess whether SUV420H1 depletion affects the proliferation of HPV-negative HSNCC cells, CCK8 and colony formation assays were pursued after siRNA-mediated knockdown of SUV420H1 in five SUV420H1-overexpressing HPV-negative HNSCC cell lines and revealed a significant reduction in cell proliferation and colony forming capacity. SUV420H1 CRISPR knockout (KO) HPV-negative HNSCC human and mouse cell lines were generated. Proliferation and colony formation assays with the SUV420H1 KO cell lines are ongoing to validate the aforementioned phenotypes. To identify oncogenic mechanism of SUV420H1, genome-wide mapping of H4K20me3 after siRNA-mediated depletion or enzymatic inhibition of SUV420H1 (A-196) was pursued using CUT&amp;RUN assays combined with RNA-seq in a SUV420H1 amplified cell line (SCC-151), and analysis is ongoing. Preliminary RNA-seq analysis revealed upregulation of a number of type I IFN response genes after siRNA-mediated knockdown of SUV420H1. In vivo mouse experiments using SUV420H1 KO mouse cell lines are planned in a syngeneic HPV-negative HNSCC flank mouse model (MOC1) with or without anti-PD-1 treatment with the goal to assess whether SUV420H1 depletion affects tumor growth and/or sensitizes cancer cells to anti-PD-1 therapy. These data provide preliminary support for the function of SUV420H1 as an oncogene in HPV-negative HNSCC. Citation Format: Arfa Moshiri, Sohyoung Kim, Marie Luff, Vassiliki Saloura. SUV420H1 depletion reveals therapeutic potential in HPV-negative head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1729.

CELSR3 is a prognostic marker in HNSCC and correlates with immune cell infiltration in the tumor microenvironment

PurposeTo look at the diagnostic value of the CELSR receptor 3 (CELSR3) gene in head and neck squamous cell carcinoma (HNSCC) and its effect on tumor immune invasion, which is important for enhancing HNSCC treatment.MethodsSeveral bioinformatics tools were employed to investigate CELSR3’s putative oncogenic pathway in HNSCC, and datasets from The Tumor Genome Atlas (TCGA), Tumor Immune Estimation Resource (TIMER), Gene Expression Profile Interaction Analysis (GEPIA) and LinkedOmics were extracted and evaluated. CELSR3 has been linked to tumor immune cell infiltration, immunological checkpoints, and immune-related genes. CELSR3’s putative roles were investigated using Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and pathway enrichment analysis. The expression level of CELSR3 in HNSCC tissues and cells was detected by RT-qPCR. The effects of CELSR3 on proliferation of HNSCC cells were detected by CCK-8 assay.ResultsCELSR3 was shown to be expressed differently in different types of cancer and normal tissues. CELSR3 gene expression was linked to pN-stage and pM-stage. Patients with high CELSR3 expression also have a well prognosis. CELSR3 expression was found to be an independent predictive factor for HNSCC in both univariate and multivariate Cox regression analyses. We discovered the functional network of CELSR3 in HNSCC using GO and KEGG analysis. CELSR3 expression levels were found to be favorably associated with immune cell infiltration levels. Furthermore, CELSR3 expression levels were significantly correlated with the expression levels of many immune molecules, such as MHC genes, immune activation genes, chemokine receptors, and chemokines. CELSR3 is highly expressed in HNSCC tissues and cells. CELSR3 overexpression significantly inhibited the proliferation of HNSCC cells. CELSR3 expression may affect the immune microenvironment and, as a result, the prognosis of HNSCC.ConclusionCELSR3 expression is elevated in HNSCC tumor tissues, and high CELSR3 expression is associated with well prognosis, which inhibited the proliferation of NHSCC cells. CELSR3 has the potential to influence tumor formation by controlling tumor-infiltrating cells in the tumor microenvironment (TME). As a result, CELSR3 may have diagnostic significance in HNSCC.

Nicotine downregulates miR-375–3p via neurotrophic tyrosine receptor kinase 2 to enhance the malignant behaviors of laryngopharyngeal squamous epithelial cells

Nicotine exposure from smoking constitutes a significant global public health concern. Furthermore, smoking represents a pivotal risk factor for head and neck squamous cell carcinoma (HNSCC). However, the influence of nicotine on HNSCC remains relatively underexplored. Our aim was to unravel the molecular mechanisms that underlie the effect of nicotine on the metastatic cascade of HNSCC. In this study, we discovered a significant association between smoking and HNSCC metastasis and prognosis. Nicotine significantly enhanced HNSCC cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) in vitro. Analysis of TCGA-HNSCC and FDEENT-HNSCC cohorts revealed reduced miR-375–3p levels in HNSCC tumor tissues, particularly among current smokers. Additionally, miR-375–3p level was strongly correlated with both lymph node metastasis and tumor stage. By downregulating miR-375–3p, nicotine promotes HNSCC cell metastasis in vitro and hematogenous metastatic capacity in vivo. Utilizing transcriptomic sequencing, molecular docking, dual-luciferase reporter assay, and fluorescence in situ hybridization (FISH), we demonstrated that miR-375–3p specifically binds to 3′ untranslated region (3’UTR) of NTRK2 mRNA. Thus, this study uncovers a novel nicotine-induced mechanism involving miR-375–3p-mediated NTRK2 targeting, which promotes HNSCC metastasis. These findings have implications for improving the prognosis of patients with HNSCC, especially in smokers.

Identification of splicing factors signature predicting prognosis risk and the mechanistic roles of novel oncogenes in HNSCC

Head and neck squamous cell carcinoma (HNSCC) is the most frequent subtype of head and neck cancer, generally with a poor prognosis and limited therapeutic options due to its highly heterogeneous malignancy. In this study, we screened functional splicing regulatory RNA binding proteins (RBPs) that were closely related with the prognosis of HNSCC patients and showed significant expression differences between HNSCC tumors and normal tissues. Based on this finding, we chose six candidate genes (HNRNPC, ZCRB1, RBM12B, SF3A2, SF3B3, and SRSF11) to generate a prognostic prediction model and validated the accuracy of the prognostic model for predicting patient survival outcomes. We found that the risk score predicted by our model can serve as an independent prognostic predictor. Notably, HNSCC tumors showing higher expression of SF3B3, HNRNPC, or ZCRB1 possessed higher risk scores in the discovered prediction model. The investigation of the underlying mechanism validated that knockdown of SF3B3, HNRNPC, and ZCRB1 separately induced a substantial impairment of HNSCC cell survival. Conversely, overexpression of each of the three genes promoted tumor cellular proliferation. High throughput RNA sequencing analysis revealed that changes in the expression of SF3B3 and HNRNPC remarkably affected alternative splicing of genes related to cell cycle regulation, whereas the depletion of ZCRB1 contributed to aberrant splicing events involving in DNA damage response. In addition, the prognostic prediction model's risk score was demonstrated to be related with the immune infiltration score. Particularly, SF3B3 has a negative correlation with CD8A expression. Therefore, our findings provide promising prognosis predictors and potential therapeutic targets for better treatment efficacy of HNSCC.

Clinical relevance and therapeutic predictive ability of hypoxia biomarkers in head and neck cancer tumour models.

Tumour hypoxia promotes poor patient outcomes, with particularly strong evidence for head and neck squamous cell carcinoma (HNSCC). To effectively target hypoxia, therapies require selection biomarkers and preclinical models that can accurately model tumour hypoxia. We established 20 patient-derived xenograft (PDX) and cell line-derived xenograft (CDX) models of HNSCC that we characterised for their fidelity to represent clinical HNSCC in gene expression, hypoxia status and proliferation and that were evaluated for their sensitivity to hypoxia-activated prodrugs (HAPs). PDX models showed greater fidelity in gene expression to clinical HNSCC than cell lines, as did CDX models relative to their paired cell lines. PDX models were significantly more hypoxic than CDX models, as assessed by hypoxia gene signatures and pimonidazole immunohistochemistry, and showed similar hypoxia gene expression to clinical HNSCC tumours. Hypoxia or proliferation status alone could not determine HAP sensitivity across our 20 HNSCC and two non-HNSCC tumour models by either tumour growth inhibition or killing of hypoxia cells in an ex vivo clonogenic assay. In summary, our tumour models provide clinically relevant HNSCC models that are suitable for evaluating hypoxia-targeting therapies; however, additional biomarkers to hypoxia are required to accurately predict drug sensitivity.

PROTAC Prodrug-Integrated Nanosensitizer for Potentiating Radiation Therapy of Cancer.

Radiation therapy (RT) is one of the primary options for clinical cancer therapy, in particular advanced head and neck squamous cell carcinoma (HNSCC). Herein, the crucial role of bromodomain-containing protein 4 (BRD4)-RAD51 associated protein 1 (RAD51AP1) axis in sensitizing RT of HNSCC is revealed. A versatile nanosensitizer (RPB7H) is thus innovatively engineered by integrating a PROteolysis TArgeting Chimeras (PROTAC) prodrug (BPA771) and hafnium dioxide (HfO2 ) nanoparticles to downregulate BRD4-RAD51AP1 pathway and sensitize HNSCC tumor to RT. Upon intravenous administration, the RPB7H nanoparticles selectively accumulate at the tumor tissue and internalize into tumor cells by recognizing neuropilin-1 overexpressed in the tumor mass. HfO2 nanoparticles enhance RT effectiveness by amplifying X-ray deposition, intensifying DNA damage, and boosting oxidative stress. Meanwhile, BPA771 can be activated by RT-induced H2 O2 secretion to degrade BRD4 and inactivate RAD51AP1, thus impeding RT-induced DNA damage repair. This versatile nanosensitizer, combined with X-ray irradiation, effectively regresses HNSCC tumor growth in a mouse model. The findings introduce a PROTAC prodrug-based radiosensitization strategy by targeting the BRD4-RAD51AP1 axis, may offer a promising avenue to augment RT and more effective HNSCC therapy.