Unravelling the Complexity of HNSCC Using Single-Cell Transcriptomics.

BACKGROUND/OBJECTIVES: Head and neck squamous cell carcinoma (HNSCC) is a highly heterogeneous and the most common form of head and neck cancer, posing significant challenges for disease management. The objective of this review is to assess the utility of single-cell RNA sequencing (scRNAseq) in addressing these challenges by enabling a detailed characterization of the tumor microenvironment (TME) at the cellular level. METHODS: This review compiles and analyzes current strategies that utilize scRNAseq and other single-cell technologies in HNSCC research. RESULTS: For HNSCC etiology, scRNAseq allows for the construction of cellular atlases, characterization of different cell types, and investigation of genes and processes involved in cancer initiation, development, and progression within the TME. In terms of HNSCC diagnosis and prognosis, the resolution offered by scRNAseq enables the identification of cell type-specific signatures, enhancing prognostic models and disease stratifiers for patient outcome assessments. Regarding HNSCC treatment, scRNAseq provides insights into cellular responses to various treatments, including radiotherapy, chemotherapy, and immunotherapy, contributing to a better understanding of treatment efficacy and patient outcomes. CONCLUSIONS: This review highlights the contributions of scRNAseq to HNSCC research, addressing its cellular and biological complexity, and emphasizes its potential for advancing research and clinical practice in other cancer types.

Transcriptomic and epigenetic landscape of nimorazole-enhanced radiochemotherapy in head and neck cancer.

BACKGROUND: Hypoxia remains a challenge for the therapeutic management of head and neck squamous cell carcinoma (HNSCC). The combination of radiotherapy with nimorazole has shown treatment benefit in HNSCC, but the precise underlying molecular mechanisms remain unclear. PURPOSE: To assess and to characterize the transcriptomic/epigenetic landscape of HNSCC tumor models showing differential therapeutic response to fractionated radiochemotherapy (RCTx) combined with nimorazole. MATERIALS/METHODS: Bulk RNA-sequencing and DNA methylation experiments were conducted using untreated and treated HNSCC xenografts after 10 fractions of RCTx with and without nimorazole. These tumor models (FaDu, SAS, Cal33, SAT and UT-SCC-45) previously showed a heterogeneous response to RCTx with nimorazole. The prognostic impact of candidate genes was assessed using clinical and gene expression data from HNSCC patients treated with primary RCTx within the DKTK-ROG. RESULTS: Nimorazole responder and non-responder tumor models showed no differences in hypoxia gene signatures However, non-responder models showed upregulation of metabolic pathways. From that, a subset of 15 differentially expressed genes stratified HNSCC patients into low and high-risk groups with distinct outcome. CONCLUSION: In the present study, we found that nimorazole non-responder models were characterized by upregulation of genes involved in Retinol metabolism and xenobiotic metabolic process pathways, which might contribute to identify mechanisms of resistance to nitroimidazole compounds and potentially expand the repertoire of therapeutic options to treat HNSCC.

Enhancing mass spectrometry imaging accessibility using convolutional autoencoders for deriving hypoxia-associated peptides from tumors.

Mass spectrometry imaging (MSI) allows to study cancer's intratumoral heterogeneity through spatially-resolved peptides, metabolites and lipids. Yet, in biomedical research MSI is rarely used for biomarker discovery. Besides its high dimensionality and multicollinearity, mass spectrometry (MS) technologies typically output mass-to-charge ratio values but not the biochemical compounds of interest. Our framework makes particularly low-abundant signals in MSI more accessible. We utilized convolutional autoencoders to aggregate features associated with tumor hypoxia, a parameter with significant spatial heterogeneity, in cancer xenograft models. We highlight that MSI captures these low-abundant signals and that autoencoders can preserve them in their latent space. The relevance of individual hyperparameters is demonstrated through ablation experiments, and the contribution from original features to latent features is unraveled. Complementing MSI with tandem MS from the same tumor model, multiple hypoxia-associated peptide candidates were derived. Compared to random forests alone, our autoencoder approach yielded more biologically relevant insights for biomarker discovery.

Epigenetic Targeting to Overcome Radioresistance in Head and Neck Cancer.

(1) Background: The sensitivity of head and neck squamous cell carcinoma (HNSCC) to ionizing radiation, among others, is determined by the number of cells with high clonogenic potential and stem-like features. These cellular characteristics are dynamically regulated in response to treatment and may lead to an enrichment of radioresistant cells with a cancer stem cell (CSC) phenotype. Epigenetic mechanisms, particularly DNA and histone methylation, are key regulators of gene-specific transcription and cellular plasticity. Therefore, we hypothesized that specific epigenetic targeting may prevent irradiation-induced plasticity and may sensitize HNSCC cells to radiotherapy. (2) Methods: We compared the DNA methylome and intracellular concentrations of tricarboxylic acid cycle metabolites in radioresistant FaDu and Cal33 cell lines with their parental controls, as well as aldehyde dehydrogenase (ALDH)-positive CSCs with negative controls. Moreover, we conducted a screen of a chemical library targeting enzymes involved in epigenetic regulation in combination with irradiation and analyzed the clonogenic potential, sphere formation, and DNA repair capacity to identify compounds with both radiosensitizing and CSC-targeting potential. (3) Results: We identified the histone demethylase inhibitor GSK-J1, which targets UTX (KDM6A) and JMJD3 (KDM6B), leading to increased H3K27 trimethylation, heterochromatin formation, and gene silencing. The clonogenic survival assay after siRNA-mediated knock-down of both genes radiosensitized Cal33 and SAS cell lines. Moreover, high KDM6A expression in tissue sections of patients with HNSCC was associated with improved locoregional control after primary (n = 137) and post-operative (n = 187) radio/chemotherapy. Conversely, high KDM6B expression was a prognostic factor for reduced overall survival. (4) Conclusions: Within this study, we investigated cellular and molecular mechanisms underlying irradiation-induced cellular plasticity, a key inducer of radioresistance, with a focus on epigenetic alterations. We identified UTX (KDM6A) as a putative prognostic and therapeutic target for HNSCC patients treated with radiotherapy.

The Role of SOX2 and SOX9 in Radioresistance and Tumor Recurrence.

Head and neck squamous cell carcinoma (HNSCC) exhibits considerable variability in patient outcome. It has been reported that SOX2 plays a role in proliferation, tumor growth, drug resistance, and metastasis in a variety of cancer types. Additionally, SOX9 has been implicated in immune tolerance and treatment failures. SOX2 and SOX9 induce treatment failure by a molecular mechanism that has not yet been elucidated. This study explores the inverse association of SOX2/SOX9 and their distinct expression in tumors, influencing the tumor microenvironment and radiotherapy responses. Through public RNA sequencing data, human biopsy samples, and knockdown cellular models, we explored the effects of inverted SOX2 and SOX9 expression. We found that patients expressing SOX2LowSOX9High showed decreased survival compared to SOX2HighSOX9Low. A survival analysis of patients stratified by radiotherapy and human papillomavirus brings additional clinical relevance. We identified a gene set signature comprising newly discovered candidate genes resulting from inverted SOX2/SOX9 expression. Moreover, the TGF-ß pathway emerges as a significant predicted contributor to the overexpression of these candidate genes. In vitro findings reveal that silencing SOX2 enhances tumor radioresistance, while SOX9 silencing enhances radiosensitivity. These discoveries lay the groundwork for further studies on the therapeutic potential of transcription factors in optimizing HNSCC treatment.

New insights into RAS in head and neck cancer.

RAS genes are known to be dysregulated in cancer for several decades, and substantial effort has been dedicated to develop agents that reduce RAS expression or block RAS activation. The recent introduction of RAS inhibitors for cancer patients highlights the importance of comprehending RAS alterations in head and neck cancer (HNC). In this regard, we examine the published findings on RAS alterations and pathway activations in HNC, and summarize their role in HNC initiation, progression, and metastasis. Specifically, we focus on the intrinsic role of mutated-RAS on tumor cell signaling and its extrinsic role in determining tumor-microenvironment (TME) heterogeneity, including promoting angiogenesis and enhancing immune escape. Lastly, we summarize the intrinsic and extrinsic role of RAS alterations on therapy resistance to outline the potential of targeting RAS using a single agent or in combination with other therapeutic agents for HNC patients with RAS-activated tumors.

Prognostic biomarkers for the response to the radiosensitizer nimorazole combined with RCTx: a pre-clinical trial in HNSCC xenografts.

BACKGROUND: Tumor hypoxia is associated with resistance to radiotherapy and chemotherapy. In head and neck squamous cell carcinoma (HNSCC), nimorazole, an oxygen mimic, combined with radiotherapy (RT) enabled to improve loco-regional control (LRC) in some patients with hypoxic tumors but it is unknown whether this holds also for radiochemotherapy (RCTx). Here, we investigated the impact of nimorazole combined with RCTx in HNSCC xenografts and explored molecular biomarkers for its targeted use. METHODS: Irradiations were performed with 30 fractions in 6 weeks combined with weekly cisplatin. Nimorazole was applied before each fraction, beginning with the first or after ten fractions. Effect of RCTx with or without addition of nimorazole was quantified as permanent local control after irradiation. For histological evaluation and targeted gene expression analysis, tumors were excised untreated or after ten fractions. Using quantitative image analysis, micromilieu parameters were determined. RESULTS: Nimorazole combined with RCTx significantly improved permanent local control in two tumor models, and showed a potential improvement in two additional models. In these four models, pimonidazole hypoxic volume (pHV) was significantly reduced after ten fractions of RCTx alone. Our results suggest that nimorazole combined with RCTx might improve TCR compared to RCTx alone if hypoxia is decreased during the course of RCTx but further experiments are warranted to verify this association. Differential gene expression analysis revealed 12 genes as potential for RCTx response. When evaluated in patients with HNSCC who were treated with primary RCTx, these genes were predictive for LRC. CONCLUSIONS: Nimorazole combined with RCTx improved local tumor control in some but not in all HNSCC xenografts. We identified prognostic biomarkers with the potential for translation to patients with HNSCC.

Activation of the EGFR/PI3K/AKT pathway limits the efficacy of trametinib treatment in head and neck cancer.

Blocking the mitogen-activated protein kinase (MAPK) pathway with the MEK1/2 inhibitor trametinib has produced promising results in patients with head and neck squamous cell carcinoma (HNSCC). In the current study, we showed that trametinib treatment leads to overexpression and activation of the epidermal growth factor receptor (EGFR) in HNSCC cell lines and patient-derived xenografts. Knockdown of EGFR improved trametinib treatment efficacy both in vitro and in vivo. Mechanistically, we demonstrated that trametinib-induced EGFR overexpression hyperactivates the phosphatidylinositol 3-kinase (PI3K)/AKT pathway. In vitro, blocking the PI3K pathway with GDC-0941 (pictilisib), or BYL719 (alpelisib), prevented AKT pathway hyperactivation and enhanced the efficacy of trametinib in a synergistic manner. In vivo, a combination of trametinib and BYL719 showed superior antitumor efficacy vs. the single agents, leading to tumor growth arrest. We confirmed our findings in a syngeneic murine head and neck cancer cell line in vitro and in vivo. Taken together, our findings show that trametinib treatment induces hyperactivation of EGFR/PI3K/AKT; thus, blocking of the EGFR/PI3K pathway is required to improve trametinib efficacy in HNSCC.

DNA-Methylome-Based Tumor Hypoxia Classifier Identifies HPV-Negative Head and Neck Cancer Patients at Risk for Locoregional Recurrence after Primary Radiochemotherapy.

PURPOSE: Tumor hypoxia is a paradigmatic negative prognosticator of treatment resistance in head and neck squamous cell carcinoma (HNSCC). The lack of robust and reliable hypoxia classifiers limits the adaptation of stratified therapies. We hypothesized that the tumor DNA methylation landscape might indicate epigenetic reprogramming induced by chronic intratumoral hypoxia. EXPERIMENTAL DESIGN: A DNA-methylome-based tumor hypoxia classifier (Hypoxia-M) was trained in the TCGA (The Cancer Genome Atlas)-HNSCC cohort based on matched assignments using gene expression-based signatures of hypoxia (Hypoxia-GES). Hypoxia-M was validated in a multicenter DKTK-ROG trial consisting of human papillomavirus (HPV)-negative patients with HNSCC treated with primary radiochemotherapy (RCHT). RESULTS: Although hypoxia-GES failed to stratify patients in the DKTK-ROG, Hypoxia-M was independently prognostic for local recurrence (HR, 4.3; P = 0.001) and overall survival (HR, 2.34; P = 0.03) but not distant metastasis after RCHT in both cohorts. Hypoxia-M status was inversely associated with CD8 T-cell infiltration in both cohorts. Hypoxia-M was further prognostic in the TCGA-PanCancer cohort (HR, 1.83; P = 0.04), underscoring the breadth of this classifier for predicting tumor hypoxia status. CONCLUSIONS: Our findings highlight an unexplored avenue for DNA methylation-based classifiers as biomarkers of tumoral hypoxia for identifying high-risk features in patients with HNSCC tumors. See related commentary by Heft Neal and Brenner, p. 2954.

A Novel Subgroup of UCHL1-Related Cancers Is Associated with Genomic Instability and Sensitivity to DNA-Damaging Treatment.

PURPOSE: Identification of molecularly-defined cancer subgroups and targeting tumor-specific vulnerabilities have a strong potential to improve treatment response and patient outcomes but remain an unmet challenge of high clinical relevance, especially in head and neck squamous cell carcinoma (HNSC). EXPERIMENTAL DESIGN: We established a UCHL1-related gene set to identify and molecularly characterize a UCHL1-related subgroup within TCGA-HNSC by integrative analysis of multi-omics data. An extreme gradient boosting model was trained on TCGA-HNSC based on GSVA scores for gene sets of the MSigDB to robustly predict UCHL1-related cancers in other solid tumors and cancer cell lines derived thereof. Potential vulnerabilities of UCHL1-related cancer cells were elucidated by an in-silico drug screening approach. RESULTS: We established a 497-gene set, which stratified the TCGA-HNSC cohort into distinct subgroups with a UCHL1-related or other phenotype. UCHL1-related HNSC were characterized by higher frequencies of genomic alterations, which was also evident for UCHL1-related cancers of other solid tumors predicted by the classification model. These data indicated an impaired maintenance of genomic integrity and vulnerability for DNA-damaging treatment, which was supported by a favorable prognosis of UCHL1-related tumors after radiotherapy, and a higher sensitivity of UCHL1-related cancer cells to irradiation or DNA-damaging compounds (e.g., Oxaliplatin). CONCLUSION: Our study established UCHL1-related cancers as a novel subgroup across most solid tumor entities with a unique molecular phenotype and DNA-damaging treatment as a specific vulnerability, which requires further proof-of-concept in pre-clinical models and future clinical trials.

Glioneuronal tumor with ATRX alteration, kinase fusion and anaplastic features (GTAKA): a molecularly distinct brain tumor type with recurrent NTRK gene fusions.

Glioneuronal tumors are a heterogenous group of CNS neoplasms that can be challenging to accurately diagnose. Molecular methods are highly useful in classifying these tumors-distinguishing precise classes from their histological mimics and identifying previously unrecognized types of tumors. Using an unsupervised visualization approach of DNA methylation data, we identified a novel group of tumors (n = 20) that formed a cluster separate from all established CNS tumor types. Molecular analyses revealed ATRX alterations (in 16/16 cases by DNA sequencing and/or immunohistochemistry) as well as potentially targetable gene fusions involving receptor tyrosine-kinases (RTK; mostly NTRK1-3) in all of these tumors (16/16; 100%). In addition, copy number profiling showed homozygous deletions of CDKN2A/B in 55% of cases. Histological and immunohistochemical investigations revealed glioneuronal tumors with isomorphic, round and often condensed nuclei, perinuclear clearing, high mitotic activity and microvascular proliferation. Tumors were mainly located supratentorially (84%) and occurred in patients with a median age of 19 years. Survival data were limited (n = 18) but point towards a more aggressive biology as compared to other glioneuronal tumors (median progression-free survival 12.5 months). Given their molecular characteristics in addition to anaplastic features, we suggest the term glioneuronal tumor with ATRX alteration, kinase fusion and anaplastic features (GTAKA) to describe these tumors. In summary, our findings highlight a novel type of glioneuronal tumor driven by different RTK fusions accompanied by recurrent alterations in ATRX and homozygous deletions of CDKN2A/B. Targeted approaches such as NTRK inhibition might represent a therapeutic option for patients suffering from these tumors.

Dynamics of CXCR4 positive circulating tumor cells in prostate cancer patients during radiotherapy.

Ablative radiotherapy is a highly efficient treatment modality for patients with metastatic prostate cancer (PCa). However, a subset of patients does not respond. Currently, this subgroup with bad prognosis cannot be identified before disease progression. We hypothesize that markers indicative of radioresistance, stemness and/or bone tropism may have a prognostic potential to identify patients profiting from metastases-directed radiotherapy. Therefore, circulating tumor cells (CTCs) were analyzed in patients with metastatic PCa (n = 24) during radiotherapy with CellSearch, multicolor flow cytometry and imaging cytometry. Analysis of copy-number alteration indicates a polyclonal CTC population that changes after radiotherapy. CTCs were found in 8 out of 24 patients (33.3%) and were associated with a shorter time to biochemical progression after radiotherapy. Whereas the total CTC count dropped after radiotherapy, a chemokine receptor CXCR4-expressing subpopulation representing 28.6% of the total CTC population remained stable up to 3 months. At once, we observed higher chemokine CCL2 plasma concentrations and proinflammatory monocytes. Additional functional analyses demonstrated key roles of CXCR4 and CCL2 for cellular radiosensitivity, tumorigenicity and stem-like potential in vitro and in vivo. Moreover, a high CXCR4 and CCL2 expression was found in bone metastasis biopsies of PCa patients. In summary, panCK+ CXCR4+ CTCs may have a prognostic potential in patients with metastatic PCa treated with metastasis-directed radiotherapy.

MEK1/2 inhibition transiently alters the tumor immune microenvironment to enhance immunotherapy efficacy against head and neck cancer.

BACKGROUND: Although the mitogen-activated protein kinases (MAPK) pathway is hyperactive in head and neck cancer (HNC), inhibition of MEK1/2 in HNC patients has not shown clinically meaningful activity. Therefore, we aimed to characterize the effect of MEK1/2 inhibition on the tumor microenvironment (TME) of MAPK-driven HNC, elucidate tumor-host interaction mechanisms facilitating immune escape on treatment, and apply rationale-based therapy combination immunotherapy and MEK1/2 inhibitor to induce tumor clearance. METHODS: Mouse syngeneic tumors and xenografts experiments were used to analyze tumor growth in vivo. Single-cell cytometry by time of flight, flow cytometry, and tissue stainings were used to profile the TME in response to trametinib (MEK1/2 inhibitor). Co-culture of myeloid-derived suppressor cells (MDSC) with CD8+ T cells was used to measure immune suppression. Overexpression of colony-stimulating factor-1 (CSF-1) in tumor cells was used to show the effect of tumor-derived CSF-1 on sensitivity to trametinib and anti-programmed death- 1 (αPD-1) in mice. In HNC patients, the ratio between CSF-1 and CD8A was measured to test the association with clinical benefit to αPD-1 and αPD-L1 treatment. RESULTS: Using preclinical HNC models, we demonstrated that treatment with trametinib delays HNC initiation and progression by reducing tumor cell proliferation and enhancing the antitumor immunity of CD8+ T cells. Activation of CD8+ T cells by supplementation with αPD-1 antibody eliminated tumors and induced an immune memory in the cured mice. Mechanistically, an early response to trametinib treatment sensitized tumors to αPD-1-supplementation by attenuating the expression of tumor-derived CSF-1, which reduced the abundance of two CSF-1R+CD11c+ MDSC populations in the TME. In contrast, prolonged treatment with trametinib abolished the antitumor activity of αPD-1, because tumor cells undergoing the epithelial to mesenchymal transition in response to trametinib restored CSF-1 expression and recreated an immune-suppressive TME. CONCLUSION: Our findings provide the rationale for testing the trametinib/αPD-1 combination in HNC and highlight the importance of sensitizing tumors to αPD-1 by using MEK1/2 to interfere with the tumor-host interaction. Moreover, we describe the concept that treatment of cancer with a targeted therapy transiently induces an immune-active microenvironment, and supplementation of immunotherapy during this time further activates the antitumor machinery to cause tumor elimination.

Cellular plasticity upon proton irradiation determines tumor cell radiosensitivity.

Proton radiotherapy has been implemented into the standard-of-care for cancer patients within recent years. However, experimental studies investigating cellular and molecular mechanisms are lacking, and prognostic biomarkers are needed. Cancer stem cell (CSC)-related biomarkers, such as aldehyde dehydrogenase (ALDH), are known to influence cellular radiosensitivity through inactivation of reactive oxygen species, DNA damage repair, and cell death. In a previous study, we found that ionizing radiation itself enriches for ALDH-positive CSCs. In this study, we analyze CSC marker dynamics in prostate cancer, head and neck cancer, and glioblastoma cells upon proton beam irradiation. We find that proton irradiation has a higher potential to target CSCs through induction of complex DNA damages, lower rates of cellular senescence, and minor alteration in histone methylation pattern compared with conventional photon irradiation. Mathematical modeling indicates differences in plasticity rates among ALDH-positive CSCs and ALDH-negative cancer cells between the two irradiation types.

Plasticity within Aldehyde Dehydrogenase-Positive Cells Determines Prostate Cancer Radiosensitivity.

Tumor heterogeneity and cellular plasticity are key determinants of tumor progression, metastatic spread, and therapy response driven by the cancer stem cell (CSC) population. Within the current study, we analyzed irradiation-induced plasticity within the aldehyde dehydrogenase (ALDH)-positive (ALDH+) population in prostate cancer. The radiosensitivity of xenograft tumors derived from ALDH+ and ALDH-negative (ALDH-) cells was determined with local tumor control analyses and demonstrated different dose-response profiles, time to relapse, and focal adhesion signaling. The transcriptional heterogeneity was analyzed in pools of 10 DU145 and PC3 cells with multiplex gene expression analyses and illustrated a higher degree of heterogeneity within the ALDH+ population that even increases upon irradiation in comparison with ALDH- cells. Phenotypic conversion and clonal competition were analyzed with fluorescence protein-labeled cells to distinguish cellular origins in competitive three-dimensional cultures and xenograft tumors. We found that the ALDH+ population outcompetes ALDH- cells and drives tumor growth, in particular upon irradiation. The observed dynamics of the cellular state compositions between ALDH+ and ALDH- cells in vivo before and after tumor irradiation was reproduced by a probabilistic Markov compartment model that incorporates cellular plasticity, clonal competition, and phenotype-specific radiosensitivities. Transcriptional analyses indicate that the cellular conversion from ALDH- into ALDH+ cells within xenograft tumors under therapeutic pressure was partially mediated through induction of the transcriptional repressor SNAI2. In summary, irradiation-induced cellular conversion events are present in xenograft tumors derived from prostate cancer cells and may be responsible for radiotherapy failure. IMPLICATIONS: The increase of ALDH+ cells with stem-like features in prostate xenograft tumors after local irradiation represents a putative cellular escape mechanism inducing tumor radioresistance.

The role of tumor acidification in aggressiveness, cell dissemination and treatment resistance of oral squamous cell carcinoma.

AIMS: To investigate the role of tumor acidification in cell behavior, migration, and treatment resistance of oral squamous cell carcinoma (OSCC). MAIN METHODS: The SCC4 and SCC25 cell lines were exposed to acidified (pH 6.8) cell culture medium for 7 days. Alternatively, a long-term acidosis was induced for 21 days. In addition, to mimic dynamic pH fluctuation of the tumor microenvironment, cells were reconditioned to neutral pH after experimental acidosis. This study assessed cell proliferation and viability by sulforhodamine B and flow cytometry. Individual and collective cell migration was analyzed by wound healing, time lapse, and transwell assays. Modifications of cell phenotype, EMT induction and stemness potential were investigated by qRT-PCR, western blot, and immunofluorescence. Finally, resistance to chemo- and radiotherapy of OSCC when exposed to acidified environmental conditions (pH 6.8) was determined. KEY FINDINGS: The exposure to an acidic microenvironment caused an initial reduction of OSCC cells viability, followed by an adaptation process. Acidic adapted cells acquired a mesenchymal-like phenotype along with increased migration and motility indexes. Moreover, tumoral extracellular acidity was capable to induce cellular stemness and to increase chemo- and radioresistance of oral cancer cells. SIGNIFICANCE: In summary, the results showed that the acidic microenvironment leads to a more aggressive and treatment resistant OSCC cell population.

High-Complexity cellular barcoding and clonal tracing reveals stochastic and deterministic parameters of radiation resistance.

It is elusive whether clonal selection of tumor cells in response to ionizing radiation (IR) is a deterministic or stochastic process. With high resolution clonal barcoding and tracking of over 400 000 HNSCC patient-derived tumor cells the clonal dynamics of tumor cells in response to IR was analyzed. Fractionated IR induced a strong selective pressure for clonal reduction which significantly exceeded uniform clonal survival probabilities indicative for a strong clone-to-clone difference within tumor cell lines. IR induced clonal reduction affected the majority of tumor cells ranging between 96% and 75% and correlated to the degree of radiation sensitivity. Survival to IR is driven by a deterministic clonal selection of a smaller population which commonly survives radiation, while increased clonogenic capacity is a result of clonal competition of cells which have been selected stochastically. A 2-fold increase in radiation resistance results in a 4-fold (P < .05) higher deterministic clonal selection showing that the ratio of these parameters is amenable to radiation sensitivity which correlates to prognostic biomarkers of HNSCC. Evidence for the existence of a rare subpopulation with an intrinsically radiation resistant phenotype commonly surviving IR was found at a frequency of 0.6% to 3.3% (P < .001, FDR 3%). With cellular barcoding we introduce a novel functional heterogeneity associated qualitative readout for tracking dynamics of clonogenic survival in response to radiation. This enables the quantification of intrinsically radiation resistant tumor cells from patient samples and reveals the contribution of stochastic and deterministic clonal selection processes in response to IR.

Establishment of a Plasticity-Associated Risk Model Based on a SOX2- and SOX9-Related Gene Set in Head and Neck Squamous Cell Carcinoma.

Recent studies highlighted SOX2 and SOX9 as key determinants for cancer-cell plasticity and demonstrated that cisplatin-induced adaptation in oral squamous cell carcinoma (SCC) is acquired by an inverse regulation of both transcription factors. However, the association between SOX2/SOX9-related genetic programs with risk factors and genetic or epigenetic alterations in primary head and neck SCC (HNSCC), and their prognostic value is largely unknown.Here, we identified differentially-expressed genes (DEG) related to SOX2 and SOX9 transcription in The Cancer Genome Atlas (TCGA)-HNSC, which enable clustering of patients into groups with distinct clinical features and survival. A prognostic risk model was established by LASSO Cox regression based on expression patterns of DEGs in TCGA-HNSC (training cohort), and was confirmed in independent HNSCC validation cohorts as well as other cancer cohorts from TCGA. Differences in the mutational landscape among risk groups of TCGA-HNSC demonstrated an enrichment of truncating NSD1 mutations for the low-risk group and elucidated DNA methylation as modulator of SOX2 expression. Gene set variation analysis (GSVA) revealed differences in several oncogenic pathways among risk groups, including upregulation of gene sets related to oncogenic KRAS signaling for the high-risk group. Finally, in silico drug screen analysis revealed numerous compounds targeting EGFR signaling with significantly lower efficacy for cancer cell lines with a higher risk phenotype, but also indicated potential vulnerabilities. IMPLICATIONS: The established risk model identifies patients with primary HNSCC, but also other cancers at a higher risk for treatment failure, who might benefit from a therapy targeting SOX2/SOX9-related gene regulatory and signaling networks.

Oct4 confers stemness and radioresistance to head and neck squamous cell carcinoma by regulating the homologous recombination factors PSMC3IP and RAD54L.

Head and neck squamous cell carcinoma (HNSCC) is often being diagnosed at an advanced stage, conferring a poor prognosis. The probability of local tumor control after radiotherapy depends on the eradication of cancer stem cells (CSCs) with activated DNA repair. This study provides evidence that the CSC-related transcription factor Oct4 contributes to HNSCC radioresistance by regulating DNA damage response and the CSC phenotype. Knockdown of Oct4 A isoform reduced self-renewal capacity in HNSCC and led to partial tumor cell radiosensitization caused by transcriptional downregulation of the cell cycle checkpoint kinases CHK1 and WEE1 and homologous recombination (HR) repair genes PSMC3IP and RAD54L. Besides, PARP inhibition with Olaparib selectively radiosensitized Oct4 A knockout, but not wild-type HNSCC cells. This finding links Oct4 A to the HR-mediated DNA repair mechanisms. In turn, knockdown of PSMC3IP and RAD54L reduced the HNSCC self-renewal capacity and clonogenic cell survival after irradiation, suggesting the interplay between DNA repair and the CSC phenotype. Similar to the effect of Oct4 knockdown, overexpression of Oct4 also resulted in significant HNSCC radiosensitization and increased DNA damage, suggesting that Oct4-dependent regulation of DNA repair depends on its fine-tuned expression. In line with this observation, HNSCC patients with high and low nuclear Oct4 expression at the invasive tumor front exhibited better loco-regional tumor control after postoperative radio(chemo)therapy compared to the intermediate expression subgroup. Thus, we found that the Oct4-driven transcriptional program plays a critical role in regulating HNSCC radioresistance, and a combination of radiotherapy with PARP inhibitors may induce synthetic lethality in Oct4-deregulated tumors.

Tyrosine Kinase c-MET as Therapeutic Target for Radiosensitization of Head and Neck Squamous Cell Carcinomas.

The receptor tyrosine kinase c-MET activates intracellular signaling and induces cell proliferation, epithelial-to-mesenchymal-transition and migration. Within the present study, we validated the prognostic value of c-MET in patients with head and neck squamous cell carcinoma (HNSCC) treated with radio(chemo)therapy using the Cancer Genome Atlas database and found an association of increased MET gene expression and protein phosphorylation with reduced disease-specific and progression-free survival. To investigate the role of c-MET-dependent radioresistance, c-MET-positive cells were purified from established HNSCC cell lines and a reduced radiosensitivity and enhanced sphere-forming potential, compared to the c-MET-depleted cell population, was found in two out of four analyzed cell lines pointing to regulatory heterogeneity. We showed that c-MET is dynamically regulated after irradiation in vitro and in vivo. Interestingly, no direct impact of c-MET on DNA damage repair was found. The therapeutic potential of eight c-MET targeting agents in combination with irradiation demonstrated variable response rates in six HNSCC cell lines. Amongst them, crizotinib, foretinib, and Pha665752 exhibited the strongest radiosensitizing effect. Kinase activity profiling showed an association of crizotinib resistance with compensatory PI3K/AKT and MAP kinase signaling. Overall, our results indicate that c-MET is conferring radioresistance in HNSCC through modulation of intracellular kinase signaling and stem-like features.