CD4+ T cell activation distinguishes response to anti-PD-L1+anti-CTLA4 therapy from anti-PD-L1 monotherapy.

Cancer patients often receive a combination of antibodies targeting programmed death-ligand 1 (PD-L1) and cytotoxic T lymphocyte antigen-4 (CTLA4). We conducted a window-of-opportunity study in head and neck squamous cell carcinoma (HNSCC) to examine the contribution of anti-CTLA4 to anti-PD-L1 therapy. Single-cell profiling of on- versus pre-treatment biopsies identified T cell expansion as an early response marker. In tumors, anti-PD-L1 triggered the expansion of mostly CD8+ T cells, whereas combination therapy expanded both CD4+ and CD8+ T cells. Such CD4+ T cells exhibited an activated T helper 1 (Th1) phenotype. CD4+ and CD8+ T cells co-localized with and were surrounded by dendritic cells expressing T cell homing factors or antibody-producing plasma cells. T cell receptor tracing suggests that anti-CTLA4, but not anti-PD-L1, triggers the trafficking of CD4+ naive/central-memory T cells from tumor-draining lymph nodes (tdLNs), via blood, to the tumor wherein T cells acquire a Th1 phenotype. Thus, CD4+ T cell activation and recruitment from tdLNs are hallmarks of early response to anti-PD-L1 plus anti-CTLA4 in HNSCC.

Methylation of histone H3 lysine 36 is a barrier for therapeutic interventions of head and neck squamous cell carcinoma.

Approximately 20% of head and neck squamous cell carcinomas (HNSCCs) exhibit reduced methylation on lysine 36 of histone H3 (H3K36me) due to mutations in histone methylase NSD1 or a lysine-to-methionine mutation in histone H3 (H3K36M). Whether such alterations of H3K36me can be exploited for therapeutic interventions is still unknown. Here, we show that HNSCC models expressing H3K36M can be divided into two groups: those that display aberrant accumulation of H3K27me3 and those that maintain steady levels of H3K27me3. The former group exhibits reduced proliferation, genome instability, and heightened sensitivity to genotoxic agents like PARP1/2 inhibitors. Conversely, H3K36M HNSCC models with constant H3K27me3 levels lack these characteristics unless H3K27me3 is elevated by DNA hypomethylating agents or inhibiting H3K27me3 demethylases KDM6A/B. Mechanistically, H3K36M reduces H3K36me by directly impeding the activities of the histone methyltransferase NSD3 and the histone demethylase LSD2. Notably, aberrant H3K27me3 levels induced by H3K36M expression are not a bona fide epigenetic mark because they require continuous expression of H3K36M to be inherited. Moreover, increased sensitivity to PARP1/2 inhibitors in H3K36M HNSCC models depends solely on elevated H3K27me3 levels and diminishing BRCA1- and FANCD2-dependent DNA repair. Finally, a PARP1/2 inhibitor alone reduces tumor burden in a H3K36M HNSCC xenograft model with elevated H3K27me3, whereas in a model with consistent H3K27me3, a combination of PARP1/2 inhibitors and agents that up-regulate H3K27me3 proves to be successful. These findings underscore the crucial balance between H3K36 and H3K27 methylation in maintaining genome instability, offering new therapeutic options for patients with H3K36me-deficient tumors.

RBM33 is a unique m6A RNA-binding protein that regulates ALKBH5 demethylase activity and substrate selectivity.

Regulation of RNA substrate selectivity of m6A demethylase ALKBH5 remains elusive. Here, we identify RNA-binding motif protein 33 (RBM33) as a previously unrecognized m6A-binding protein that plays a critical role in ALKBH5-mediated mRNA m6A demethylation of a subset of mRNA transcripts by forming a complex with ALKBH5. RBM33 recruits ALKBH5 to its m6A-marked substrate and activates ALKBH5 demethylase activity through the removal of its SUMOylation. We further demonstrate that RBM33 is critical for the tumorigenesis of head-neck squamous cell carcinoma (HNSCC). RBM33 promotes autophagy by recruiting ALKBH5 to demethylate and stabilize DDIT4 mRNA, which is responsible for the oncogenic function of RBM33 in HNSCC cells. Altogether, our study uncovers the mechanism of selectively demethylate m6A methylation of a subset of transcripts during tumorigenesis that may explain demethylation selectivity in other cellular processes, and we showed its importance in the maintenance of tumorigenesis of HNSCC.

The periosteum provides a stromal defence against cancer invasion into the bone.

The periosteum is the layer of cells that covers nearly the entire surface of every bone. Upon infection, injury or malignancy the bone surface undergoes new growth-the periosteal reaction-but the mechanism and physiological role of this process remain unknown1,2. Here we show that the periosteal reaction protects against cancer invasion into the bone. Histological analyses of human lesions of head and neck squamous cell carcinomas (HNSCCs) show that periosteal thickening occurs in proximity to the tumour. We developed a genetically dissectible mouse model of HNSCC and demonstrate that inducible depletion of periosteal cells accelerates cancerous invasion of the bone. Single-cell RNA sequencing reveals that expression of the gene encoding the protease inhibitor TIMP1 is markedly increased in the periosteum at the pre-invasive stage. This increase is due to upregulation of HIF1α expression in the tumour microenvironment, and increased TIMP1 inactivates matrix-degrading proteases, promoting periosteal thickening to inhibit cancer invasion. Genetic deletion of Timp1 impairs periosteal expansion, exacerbating bone invasion and decreasing survival in tumour-bearing mice. Together, these data show that the periosteal reaction may act as a functional stromal barrier against tumour progression, representing a unique example of tissue immunity mediated by stromal cells.

Tissue-resident memory features are linked to the magnitude of cytotoxic T cell responses in human lung cancer.

Therapies that boost the anti-tumor responses of cytotoxic T lymphocytes (CTLs) have shown promise; however, clinical responses to the immunotherapeutic agents currently available vary considerably, and the molecular basis of this is unclear. We performed transcriptomic profiling of tumor-infiltrating CTLs from treatment-naive patients with lung cancer to define the molecular features associated with the robustness of anti-tumor immune responses. We observed considerable heterogeneity in the expression of molecules associated with activation of the T cell antigen receptor (TCR) and of immunological-checkpoint molecules such as 4-1BB, PD-1 and TIM-3. Tumors with a high density of CTLs showed enrichment for transcripts linked to tissue-resident memory cells (TRM cells), such as CD103, and CTLs from CD103hi tumors displayed features of enhanced cytotoxicity. A greater density of TRM cells in tumors was predictive of a better survival outcome in lung cancer, and this effect was independent of that conferred by CTL density. Here we define the 'molecular fingerprint' of tumor-infiltrating CTLs and identify potentially new targets for immunotherapy.

Immune Landscape of Viral- and Carcinogen-Driven Head and Neck Cancer.

Head and neck squamous cell carcinoma (HNSCC) arises through exposure to environmental carcinogens or malignant transformation by human papillomavirus (HPV). Here, we assessed the transcriptional profiles of 131,224 single cells from peripheral and intra-tumoral immune populations from patients with HPV- and HPV+ HNSCC and healthy donors. Immune cells within tumors of HPV- and HPV+ HNSCC displayed a spectrum of transcriptional signatures, with helper CD4+ T cells and B cells being relatively divergent and CD8+ T cells and CD4+ regulatory T cells being relatively similar. Transcriptional results were contextualized through multispectral immunofluorescence analyses and evaluating putative cell-cell communication based on spatial proximity. These analyses defined a gene expression signature associated with CD4+ T follicular helper cells that is associated with longer progression-free survival in HNSCC patients. The datasets and analytical approaches herein provide a resource for the further study of the impact of immune cells on viral- and carcinogen-induced cancers.

Genomic signature of Fanconi anaemia DNA repair pathway deficiency in cancer.

Fanconi anaemia (FA), a model syndrome of genome instability, is caused by a deficiency in DNA interstrand crosslink repair resulting in chromosome breakage1-3. The FA repair pathway protects against endogenous and exogenous carcinogenic aldehydes4-7. Individuals with FA are hundreds to thousands fold more likely to develop head and neck (HNSCC), oesophageal and anogenital squamous cell carcinomas8 (SCCs). Molecular studies of SCCs from individuals with FA (FA SCCs) are limited, and it is unclear how FA SCCs relate to sporadic HNSCCs primarily driven by tobacco and alcohol exposure or infection with human papillomavirus9 (HPV). Here, by sequencing genomes and exomes of FA SCCs, we demonstrate that the primary genomic signature of FA repair deficiency is the presence of high numbers of structural variants. Structural variants are enriched for small deletions, unbalanced translocations and fold-back inversions, and are often connected, thereby forming complex rearrangements. They arise in the context of TP53 loss, but not in the context of HPV infection, and lead to somatic copy-number alterations of HNSCC driver genes. We further show that FA pathway deficiency may lead to epithelial-to-mesenchymal transition and enhanced keratinocyte-intrinsic inflammatory signalling, which would contribute to the aggressive nature of FA SCCs. We propose that the genomic instability in sporadic HPV-negative HNSCC may arise as a result of the FA repair pathway being overwhelmed by DNA interstrand crosslink damage caused by alcohol and tobacco-derived aldehydes, making FA SCC a powerful model to study tumorigenesis resulting from DNA-crosslinking damage.

METTL3 and N6-Methyladenosine Promote Homologous Recombination-Mediated Repair of DSBs by Modulating DNA-RNA Hybrid Accumulation.

Double-strand breaks (DSBs) are the most deleterious DNA lesions, which, if left unrepaired, may lead to genome instability or cell death. Here, we report that, in response to DSBs, the RNA methyltransferase METTL3 is activated by ATM-mediated phosphorylation at S43. Phosphorylated METTL3 is then localized to DNA damage sites, where it methylates the N6 position of adenosine (m6A) in DNA damage-associated RNAs, which recruits the m6A reader protein YTHDC1 for protection. In this way, the METTL3-m6A-YTHDC1 axis modulates accumulation of DNA-RNA hybrids at DSBs sites, which then recruit RAD51 and BRCA1 for homologous recombination (HR)-mediated repair. METTL3-deficient cells display defective HR, accumulation of unrepaired DSBs, and genome instability. Accordingly, depletion of METTL3 significantly enhances the sensitivity of cancer cells and murine xenografts to DNA damage-based therapy. These findings uncover the function of METTL3 and YTHDC1 in HR-mediated DSB repair, which may have implications for cancer therapy.

An update on the immune landscape in lung and head and neck cancers.

Immunotherapy has dramatically changed the treatment landscape for patients with cancer. Programmed death-ligand 1/programmed death-1 checkpoint inhibitors have been in the forefront of this clinical revolution. Currently, there are 6 US Food and Drug Administration-approved checkpoint inhibitors for approximately 18 different histologic types of cancer. Lung cancer and head and neck squamous cell carcinoma (HNSCC) are 2 diseases that have led the way in the development of immunotherapy. Atezolizumab, durvalumab, nivolumab, and pembrolizumab are all currently used as part of standard-of-care treatment for different stages of lung cancer. Similarly, nivolumab and pembrolizumab have US regulatory approval as treatment for advanced metastatic HNSCC. This is significant because lung cancer represents the most common and most fatal cancer globally, and HNSCC is the sixth most common. Currently, most of the approvals for the use of immunotherapy agents are for patients diagnosed in the metastatic setting. However, research is ongoing to evaluate these drugs in earlier stage disease. There is plausible biological rationale to expect that pharmacologic activation of the immune system will be effective for early-stage and smaller tumors. In addition, selecting patients who are more likely to respond to immunotherapy and understanding why resistance develops are crucial areas of ongoing research. The objective of this review was to provide an overview of the current immune landscape and future directions in lung cancer and HNSCC.

TM4SF19 controls GABP-dependent YAP transcription in head and neck cancer under oxidative stress conditions.

Tobacco and alcohol are risk factors for human papillomavirus-negative head and neck squamous cell carcinoma (HPV- HNSCC), which arises from the mucosal epithelium of the upper aerodigestive tract. Notably, despite the mutagenic potential of smoking, HPV- HNSCC exhibits a low mutational load directly attributed to smoking, which implies an undefined role of smoking in HPV- HNSCC. Elevated YAP (Yes-associated protein) mRNA is prevalent in HPV- HNSCC, irrespective of the YAP gene amplification status, and the mechanism behind this upregulation remains elusive. Here, we report that oxidative stress, induced by major risk factors for HPV- HNSCC such as tobacco and alcohol, promotes YAP transcription via TM4SF19 (transmembrane 4 L six family member 19). TM4SF19 modulates YAP transcription by interacting with the GABP (Guanine and adenine-binding protein) transcription factor complex. Mechanistically, oxidative stress induces TM4SF19 dimerization and topology inversion in the endoplasmic reticulum membrane, which in turn protects the GABPß1 subunit from proteasomal degradation. Conversely, depletion of TM4SF19 impairs the survival, proliferation, and migration of HPV- HNSCC cells, highlighting the potential therapeutic relevance of targeting TM4SF19. Our findings reveal the roles of the key risk factors of HPV- HNSCC in tumor development via oxidative stress, offering implications for upcoming therapeutic approaches in HPV- HNSCC.

A druggable cascade links methionine metabolism to epigenomic reprogramming in squamous cell carcinoma.

Upper aerodigestive squamous cell carcinoma (UASCC) is a common and aggressive malignancy with few effective therapeutic options. Here, we investigate amino acid metabolism in this cancer, surprisingly noting that UASCC exhibits the highest methionine level across all human cancers, driven by its transporter LAT1. We show that LAT1 is also expressed at the highest level in UASCC, transcriptionally activated by UASCC-specific promoter and enhancers, which are directly coregulated by SCC master regulators TP63/KLF5/SREBF1. Unexpectedly, unbiased bioinformatic screen identifies EZH2 as the most significant target downstream of the LAT1-methionine pathway, directly linking methionine metabolism to epigenomic reprogramming. Importantly, this cascade is indispensable for the survival and proliferation of UASCC patient-derived tumor organoids. In addition, LAT1 expression is closely associated with cellular sensitivity to inhibition of the LAT1-methionine-EZH2 axis. Notably, this unique LAT1-methionine-EZH2 cascade can be targeted effectively by either pharmacological approaches or dietary intervention in vivo. In summary, this work maps a unique mechanistic cross talk between epigenomic reprogramming with methionine metabolism, establishes its biological significance in the biology of UASCC, and identifies a unique tumor-specific vulnerability which can be exploited both pharmacologically and dietarily.

ETV4/NSUN2 Axis modulates aerobic glycolysis and malignancy in HSCC.

This study delves into the molecular intricacies of hypopharyngeal squamous cell carcinoma (HSCC), specifically focusing on the pivotal role played by ETS translocation variant 4 (ETV4) in aerobic glycolysis. The objective is to uncover new targets for early diagnosis and treatment of HSCC. ETV4 expression in HSCC tissues was rigorously examined, revealing its association with patient survival. Through comprehensive experimentation, we demonstrated that ETV4 activation promotes HSCC cell proliferation and invasion while inhibiting apoptosis. Furthermore, in vivo experiments confirmed the tumor-promoting effect of ETV4 activation. The study elucidated the binding of ETV4 to the NSUN2 promoter and its influence on PKM2 expression, thereby regulating glycolysis and cellular functions in HSCC.

Engineering Dimeric EGFR-directed IgA Antibodies Reveals a Central Role of CD147 during Neutrophil-mediated Tumor Cell Killing of Head and Neck Squamous Cancer Cells.

Human IgA Abs engage neutrophils for cancer immunotherapy more effectively than IgG Abs. Previous studies demonstrated that engineering approaches improved biochemical and functional properties. In this study, we report a novel, to our knowledge, IgA2 Ab against the epidermal growth factor receptor generated by protein engineering and polymerization. The resulting molecule demonstrated a covalent linkage of L and H chains and an effective polymerization by the joining chain. The engineered dimer outperformed its monomeric variant in functional experiments on Fab-mediated modes of action and binding to the Fc receptor. The capacity to engage neutrophils for Ab-dependent cell-mediated cytotoxicity (ADCC) of adherent growing target cancer cells was cell line dependent. Although the engineered dimer displayed a long-term efficacy against the vulva carcinoma cell line A431, there was a notable in-efficacy against human papillomavirus (HPV)- head and neck squamous cell carcinoma (HNSCC) cell lines. However, the highly engineered IgA Abs triggered a neutrophil-mediated cytotoxicity against HPV+ HNSCC cell lines. Short-term ADCC efficacy correlated with the target cells' epidermal growth factor receptor expression and the ability of cancer cell-conditioned media to enhance the CD147 surface level on neutrophils. Notably, the HPV+ HNSCC cell lines demonstrated a significant increment in releasing soluble CD147 and a reduced induction of membranous CD147 on neutrophils compared with HPV- cells. Although membranous CD147 on neutrophils may impair proper IgA-Fc receptor binding, soluble CD147 enhanced the IgA-neutrophil-mediated ADCC in a dose-dependent manner. Thus, engineering IgA Abs and impedance-based ADCC assays provided valuable information regarding the target-effector cell interaction and identified CD147 as a putative critical parameter for neutrophil-mediated cytotoxicity.

Identification of Regulatory RNA-Binding Proteins Associated with Immune Infiltration in Laryngeal Squamous Cell Carcinoma.

We analyzed bulk RNA sequencing and single-cell RNA sequencing (scRNA-seq) data to identify alternative splicing (AS) events and regulatory RNA-binding proteins (RBPs) associated with immune infiltration in human laryngeal squamous cell carcinoma (LSCC). Whole-transcriptome sequencing data of 20 human laryngeal cancer and paracancerous tissues were downloaded from the Gene Expression Omnibus public database, using newly published splicing-site usage variation analysis software to obtain highly conserved regulated AS (RAS) events, and scientific reverse convolution algorithm analysis was used to identify significantly different immune cells and perform a correlation analysis between the two. The software package edgeR was used to identify differentially expressed RBPs and the immune infiltration-related LSCC-RAS they may regulate. Finally, we present the expression profiles and survival curves of 117 human laryngeal cancer samples from The Cancer Genome Atlas dataset for the identified RBPs and LSCC-RAS. We also downloaded the gene set enrichment 150321 scRNA-seq data for two human LSCC tissue samples. The RBP expression pattern and the expression of prophase RBP genes were analyzed in different LSCC cell populations. RNA-binding motif protein 47 (RBM47) and filamin A, as well as the RBP-RAS events that were screened in both the fibulin 2 and fibronectin 1 genes, were all significantly associated with the prognosis, and the RBM47 gene was upregulated in myeloid cells. Because the prognosis was significantly associated with two RBP regulators and two LSCC-RAS events, they may be critical regulators of immune cell survival during laryngeal cancer progression, and RBM47 may regulate macrophage-associated AS and affect immunity.

Galectin-3-Binding Protein Inhibits Extracellular Heparan 6-O-Endosulfatase Sulf-2.

Human extracellular 6-O-endosulfatases Sulf-1 and Sulf-2 are the only enzymes that post-synthetically alter the 6-O sulfation of heparan sulfate proteoglycans (HSPG), which regulates interactions of HSPG with many proteins. Oncogenicity of Sulf-2 in different cancers has been documented, and we have shown that Sulf-2 is associated with poor survival outcomes in head and neck squamous cell carcinoma (HNSCC). Despite its importance, limited information is available on direct protein-protein interactions of the Sulf-2 protein in the tumor microenvironment. In this study, we used monoclonal antibody (mAb) affinity purification and mass spectrometry to identify galectin-3-binding protein (LG3BP) as a highly specific binding partner of Sulf-2 in the conditioned media of HNSCC cell lines. We validated their direct interaction in vitro using recombinant proteins and have shown that the chondroitin sulfate (CS) covalently bound to the Sulf-2 influences the binding to LG3BP. We confirmed the importance of the CS chain for the interaction by generating a mutant Sulf-2 protein that lacks the CS. Importantly, we have shown that the LG3BP inhibits Sulf-2 activity in vitro in a concentration-dependent manner. As a consequence, the addition of LG3BP to a spheroid cell culture inhibited the invasion of the HNSCC cells into Matrigel. Thus, Sulf-2 interaction with LG3BP may regulate the physiological activity of the Sulf-2 enzyme as well as its activity in the tumor microenvironment.

Noncanonical HPV carcinogenesis drives radiosensitization of head and neck tumors.

We analyzed transcriptional data from 104 HPV+ (Human papillomavirus) HNSCC (head and neck squamous cell carcinoma) tumors together with two publicly available sources to identify highly robust transcriptional programs (modules) which could be detected consistently despite heterogeneous sequencing and quantification methodologies. Among 22 modules identified, we found a single module that naturally subclassifies HPV+ HNSCC tumors based on a bimodal pattern of gene expression, clusters all atypical features of HPV+ HNSCC biology into a single subclass, and predicts patient outcome in four independent cohorts. The subclass-defining gene set was strongly correlated with Nuclear factor kappa B (NF-κB) target expression. Tumors with high expression of this NF-κB module were rarely associated with activating PIK3CA alterations or viral integration, and also expressed higher levels of HPHPV E2 and had decreased APOBEC mutagenesis. Alternatively, they harbored inactivating alterations of key regulators of NF-κB, TNF receptor associated factor 3 (TRAF3), and cylindromatosis (CYLD), as well as retinoblastoma protein (RB1). HPV+ HNSCC cells in culture with experimental depletion of TRAF3 or CYLD displayed increased expression of the subclass-defining genes, as well as robust radio-sensitization, thus recapitulating both the tumor transcriptional state and improved treatment response observed in patient data. Across all gene sets investigated, methylation to expression correlations were the strongest for the subclass-defining, NF-κB-related genes. Increased tumor-infiltrating CD4+ T cells and increased Estrogen receptors alpha (ERα) expression were identified in NF-κB active tumors. Based on the relatively high rates of cure in HPV+ HNSCC, deintensification of therapy to reduce treatment-related morbidity is being studied at many institutions. Tumor subclassification based on oncogenic subtypes may help guide the selection of therapeutic intensity or modality for patients with HPV+ HNSCC.

The SRC family kinase inhibitor NXP900 demonstrates potent antitumor activity in squamous cell carcinomas.

NXP900 is a selective and potent SRC family kinase (SFK) inhibitor, currently being dosed in a phase 1 clinical trial, that locks SRC in the "closed" conformation, thereby inhibiting both kinase-dependent catalytic activity and kinase-independent functions. In contrast, several multi-targeted kinase inhibitors that inhibit SRC, including dasatinib and bosutinib, bind their target in the active "open" conformation, allowing SRC and other SFKs to act as a scaffold to promote tumorigenesis through non-catalytic functions. NXP900 exhibits a unique target selectivity profile with sub-nanomolar activity against SFK members over other kinases. This results in highly potent and specific SFK pathway inhibition. Here, we demonstrate that esophageal squamous cell carcinomas and head and neck squamous cell carcinomas are exquisitely sensitive to NXP900 treatment in cell culture and in vivo, and we identify a patient population that could benefit from treatment with NXP900.

Identification of cell types in multiplexed in situ images by combining protein expression and spatial information using CELESTA.

Advances in multiplexed in situ imaging are revealing important insights in spatial biology. However, cell type identification remains a major challenge in imaging analysis, with most existing methods involving substantial manual assessment and subjective decisions for thousands of cells. We developed an unsupervised machine learning algorithm, CELESTA, which identifies the cell type of each cell, individually, using the cell's marker expression profile and, when needed, its spatial information. We demonstrate the performance of CELESTA on multiplexed immunofluorescence images of colorectal cancer and head and neck squamous cell carcinoma (HNSCC). Using the cell types identified by CELESTA, we identify tissue architecture associated with lymph node metastasis in HNSCC, and validate our findings in an independent cohort. By coupling our spatial analysis with single-cell RNA-sequencing data on proximal sections of the same specimens, we identify cell-cell crosstalk associated with lymph node metastasis, demonstrating the power of CELESTA to facilitate identification of clinically relevant interactions.

SWEET: a single-sample network inference method for deciphering individual features in disease.

Recently, extracting inherent biological system information (e.g. cellular networks) from genome-wide expression profiles for developing personalized diagnostic and therapeutic strategies has become increasingly important. However, accurately constructing single-sample networks (SINs) to capture individual characteristics and heterogeneity in disease remains challenging. Here, we propose a sample-specific-weighted correlation network (SWEET) method to model SINs by integrating the genome-wide sample-to-sample correlation (i.e. sample weights) with the differential network between perturbed and aggregate networks. For a group of samples, the genome-wide sample weights can be assessed without prior knowledge of intrinsic subpopulations to address the network edge number bias caused by sample size differences. Compared with the state-of-the-art SIN inference methods, the SWEET SINs in 16 cancers more likely fit the scale-free property, display higher overlap with the human interactomes and perform better in identifying three types of cancer-related genes. Moreover, integrating SWEET SINs with a network proximity measure facilitates characterizing individual features and therapy in diseases, such as somatic mutation, mut-driver and essential genes. Biological experiments further validated two candidate repurposable drugs, albendazole for head and neck squamous cell carcinoma (HNSCC) and lung adenocarcinoma (LUAD) and encorafenib for HNSCC. By applying SWEET, we also identified two possible LUAD subtypes that exhibit distinct clinical features and molecular mechanisms. Overall, the SWEET method complements current SIN inference and analysis methods and presents a view of biological systems at the network level to offer numerous clues for further investigation and clinical translation in network medicine and precision medicine.

Targeting ENPP1 depletion may be a promising therapeutic strategy for treating oral squamous cell carcinoma via cytotoxic autophagy-related apoptosis.

ENPP1 depletion closely related with modulation immunotherapy of several types of cancer. However, the role of ENPP1 correlation with autophagy in oral squamous cell carcinoma (OSCC) pathogenesis remain unknown. In this study, effects of ENPP1 on OSCC cells in vitro were examined by cell proliferation assay, transwell chamber assay, flow cytometry analysis and shRNA technique. Cellular key proteins related to cell autophagy and apoptosis were evaluated by Western blot and immunofluorescent staining. Moreover, functions of ENPP1 on OSCC process were observed in nude mouse model. We reported that overexpression of ENPP1 promote the growth of OSCC cell xenografts in nude mouse model. In contrast, ENPP1 downregulation significantly inhibits OSCC cancer growth and induces apoptosis both in vitro and in vivo, which are preceded by cytotoxic autophagy. ENPP1downregulation induces a robust accumulation of autophagosomes, increases LC3B-II and decreases SQSTM1/p62 in ENPP1-shRNA-treated cells and xenografts. Mechanistic studies show that ENPP1 downregulation increases PRKAA1 phosphorylation leading to ULK1 activation. AMPK-inhibition abrogates ENPP1 downregulation-induced ULK1-activation, LC3B-turnover and SQSTM1/p62-degradation while AMPK-activation potentiates it's effects. Collectively, these data uncover that ENPP1 downregulation induces autophagic cell death in OSCC cancer, which may provide a potential therapeutic target for the treatment of OSCC.