Expanding the adenovirus toolbox: reporter viruses for studying the dynamics of human adenovirus replication.

To streamline standard virological assays, we developed a suite of nine fluorescent or bioluminescent replication competent human species C5 adenovirus reporter viruses that mimic their parental wild-type counterpart. These reporter viruses provide a rapid and quantitative readout of various aspects of viral infection and replication based on EGFP, mCherry, or NanoLuc measurement. Moreover, they permit real-time non-invasive measures of viral load, replication dynamics, and infection kinetics over the entire course of infection, allowing measurements that were not previously possible. This suite of replication competent reporter viruses increases the ease, speed, and adaptability of standard assays and has the potential to accelerate multiple areas of human adenovirus research.IMPORTANCEIn this work, we developed a versatile toolbox of nine HAdV-C5 reporter viruses and validated their functions in cell culture. These reporter viruses provide a rapid and quantitative readout of various aspects of viral infection and replication based on EGFP, mCherry, or NanoLuc measurement. The utility of these reporter viruses could also be extended for use in 3D cell culture, organoids, live cell imaging, or animal models, and provides a conceptual framework for the development of new reporter viruses representing other clinically relevant HAdV species.

Human papillomavirus E7 oncoprotein targets RNF168 to hijack the host DNA damage response.

High-risk human papillomaviruses (HR-HPVs) promote cervical cancer as well as a subset of anogenital and head and neck cancers. Due to their limited coding capacity, HPVs hijack the host cell's DNA replication and repair machineries to replicate their own genomes. How this host-pathogen interaction contributes to genomic instability is unknown. Here, we report that HPV-infected cancer cells express high levels of RNF168, an E3 ubiquitin ligase that is critical for proper DNA repair following DNA double-strand breaks, and accumulate high numbers of 53BP1 nuclear bodies, a marker of genomic instability induced by replication stress. We describe a mechanism by which HPV E7 subverts the function of RNF168 at DNA double-strand breaks, providing a rationale for increased homology-directed recombination in E6/E7-expressing cervical cancer cells. By targeting a new regulatory domain of RNF168, E7 binds directly to the E3 ligase without affecting its enzymatic activity. As RNF168 knockdown impairs viral genome amplification in differentiated keratinocytes, we propose that E7 hijacks the E3 ligase to promote the viral replicative cycle. This study reveals a mechanism by which tumor viruses reshape the cellular response to DNA damage by manipulating RNF168-dependent ubiquitin signaling. Importantly, our findings reveal a pathway by which HPV may promote the genomic instability that drives oncogenesis.

Inhibition of Human Adenovirus Replication by the Importin α/ß1 Nuclear Import Inhibitor Ivermectin.

Human adenoviruses (HAdV) are ubiquitous within the human population and comprise a significant burden of respiratory illnesses worldwide. Pediatric and immunocompromised individuals are at particular risk for developing severe disease; however, no approved antiviral therapies specific to HAdV exist. Ivermectin is an FDA-approved broad-spectrum antiparasitic drug that also exhibits antiviral properties against a diverse range of viruses. Its proposed function is inhibiting the classical protein nuclear import pathway mediated by importin-α (Imp-α) and -ß1 (Imp-ß1). Many viruses, including HAdV, rely on this host pathway for transport of viral proteins across the nuclear envelope. In this study, we show that ivermectin inhibits HAdV-C5 early gene transcription, early and late protein expression, genome replication, and production of infectious viral progeny. Similarly, ivermectin inhibits genome replication of HAdV-B3, a clinically important pathogen responsible for numerous recent outbreaks. Mechanistically, we show that ivermectin disrupts binding of the viral E1A protein to Imp-α without affecting the interaction between Imp-α and Imp-ß1. Our results further extend ivermectin's broad antiviral activity and provide a mechanistic underpinning for its mode of action as an inhibitor of cellular Imp-α/ß1-mediated nuclear import.IMPORTANCE Human adenoviruses (HAdVs) represent a ubiquitous and clinically important pathogen without an effective antiviral treatment. HAdV infections typically cause mild symptoms; however, individuals such as children, those with underlying conditions, and those with compromised immune systems can develop severe disseminated disease. Our results demonstrate that ivermectin, an FDA-approved antiparasitic agent, is effective at inhibiting replication of several HAdV types in vitro This is in agreement with the growing body of literature suggesting ivermectin has broad antiviral activity. This study expands our mechanistic knowledge of ivermectin by showing that ivermectin targets the ability of importin-α (Imp-α) to recognize nuclear localization sequences, without effecting the Imp-α/ß1 interaction. These data also exemplify the applicability of targeting host factors upon which viruses rely as a viable antiviral strategy.

A controlled trial of HNSCC patient-derived xenografts reveals broad efficacy of PI3Kα inhibition in controlling tumor growth.

Head and neck squamous cell carcinomas (HNSCCs) frequently harbor alterations in the PI3K/AKT/mTOR signaling axis, particularly in the PIK3CA gene. PI3K-targeted agents have therefore gained considerable preclinical and clinical interest as emerging therapies for HNSCC. Identification of predictive biomarkers of response would advance the clinical application of PI3K-targeted drugs for patients, in order to achieve maximal benefit. To date, studies of drug biomarkers have largely focused on screening cell lines, with much more limited in vivo testing, usually only as validation. This approach has rarely enabled accurate predictions of clinical efficacy. Recently, clinical trials of PDX models (PDX clinical trials) have been introduced as a preclinical approach to interrogate interpatient response heterogeneity. Already, PDX clinical trial responses have been demonstrated to correlate closely with patient outcomes. Here, using both an HNSCC specific, 28-cell line panel and a PDX clinical trial of 80 xenografts derived from 20 unique HNSCC tumors, we systematically examine patterns of response to PI3K inhibition in HNSCC. We find EGFR, AKT1 and CSMD1 copy number aberrations, but not PIK3CA mutations, to be associated with responsiveness to PI3K-targeted drugs. Further, we reveal PI3Kα inhibition to be almost globally tumoristatic in HNSCC xenografts regardless of PIK3CA mutational status, emphasizing its potential as a stabilizing neoadjuvant therapy for HNSCC patients.

Mimicry of Cellular A Kinase-Anchoring Proteins Is a Conserved and Critical Function of E1A across Various Human Adenovirus Species.

The E1A proteins of the various human adenovirus (HAdV) species perform the critical task of converting an infected cell into a setting primed for virus replication. While E1A proteins differ in both sequence and mechanism, the evolutionary pressure on viruses with limited coding capacity ensures that these proteins often have significant overlap in critical functions. HAdV-5 E1A is known to use mimicry to rewire cyclic AMP (cAMP) signaling by decoupling protein kinase A (PKA) from cellular A kinase-anchoring proteins (AKAPs) and utilizing PKA to its own advantage. We show here that E1As from other species of HAdV also possess this viral AKAP (vAKAP) function and examine how they manipulate PKA. E1A from most species of HAdV examined contain a small AKAP-like motif in their N terminus which targets the docking-dimerization domain of PKA as the binding interface for a conserved protein-protein interaction. This motif is also responsible for an E1A-mediated relocalization of PKA regulatory subunits from the cytoplasm into the nucleus, with species-specific E1A proteins having preference for one particular isoform of PKA subunit over another. Importantly, we showed that these newly characterized vAKAPs can integrate into cAMP-responsive transcription as well as contribute to viral genome replication and infectious progeny production for several distinct HAdV species.IMPORTANCE These data enhance the mechanistic knowledge on how HAdV E1A manipulates cellular PKA to benefit infection. The work establishes that mimicry of AKAPs and subversion of PKA-mediated cAMP signaling are conserved features for numerous human adenoviruses. This study also highlights the molecular determinants conferring selective protein-protein interactions between distinct PKA regulatory subunits and the different E1A proteins of these viruses. Additionally, it further emphasizes the utility of using viral proteins like E1A as tools for studying the molecular biology of cellular regulatory pathways.

Sweet DREAMs for Hippo.

The Hippo pathway coordinates organ size and cell proliferation. The retinoblastoma family of proteins regulates progression through the G0/G1 phase of the cell cycle. Disruption of either pathway contributes to cancer formation. Three recent studies in Genes & Development reveal how cellular proliferation is coordinated between these pathways. Here we discuss the implications of these studies and the new questions that they raise.

Inhibition of androgen receptor transactivation function by adenovirus type 12 E1A undermines prostate cancer cell survival.

BACKGROUND: Mutations or truncation of the ligand-binding domain (LBD) of androgen receptor (AR) underlie treatment resistance for prostate cancer (PCa). Thus, targeting the AR N-terminal domain (NTD) could overcome such resistance. METHODS: Luciferase reporter assays after transient transfection of various DNA constructs were used to assess effects of E1A proteins on AR-mediated transcription. Immunofluorescence microscopy and subcellular fractionation were applied to assess intracellular protein localization. Immunoprecipitation and mammalian two-hybrid assays were used to detect protein-protein interactions. qRT-PCR was employed to determine RNA levels. Western blotting was used to detect protein expression in cells. Effects of adenoviruses on prostate cancer cell survival were evaluated with CellTiter-Glo assays. RESULTS: Adenovirus 12 E1A (E1A12) binds specifically to the AR. Interestingly, the full-length E1A12 (266 aa) preferentially binds to full-length AR, while the small E1A12 variant (235 aa) interacts more strongly with AR-V7. E1A12 promotes AR nuclear translocation, likely through mediating intramolecular AR NTD-LBD interactions. In the nucleus, AR and E1A12 co-expression in AR-null PCa cells results in E1A12 redistribution from nuclear foci containing CBX4 (also known as Pc2), suggesting a preferential AR-E1A12 interaction over other E1A12 interactors. E1A12 represses AR-mediated transcription in reporter gene assays and endogenous AR target genes such as ATAD2 and MYC in AR-expressing PCa cells. AR-expressing PCa cells are more sensitive to death induced by a recombinant adenovirus expressing E1A12 (Ad-E1A12) than AR-deficient PCa cells, which could be attributed to the increased viral replication promoted by androgen stimulation. Targeting the AR by E1A12 promotes apoptosis in PCa cells that express the full-length AR or C-terminally truncated AR variants. Importantly, inhibition of mTOR signaling that blocks the expression of anti-apoptotic proteins markedly augments Ad-E1A12-induced apoptosis of AR-expressing cells. Mechanistically, Ad-E1A12 infection triggers apoptotic response while activating the PI3K-AKT-mTOR signaling axis; thus, mTOR inhibition enhances apoptosis in AR-expressing PCa cells infected by Ad-E1A12. CONCLUSION: Ad12 E1A inhibits AR-mediated transcription and suppresses PCa cell survival, suggesting that targeting the AR by E1A12 might have therapeutic potential for treating advanced PCa with heightened AR signaling.

Functional and Structural Mimicry of Cellular Protein Kinase A Anchoring Proteins by a Viral Oncoprotein.

The oncoproteins of the small DNA tumor viruses interact with a plethora of cellular regulators to commandeer control of the infected cell. During infection, adenovirus E1A deregulates cAMP signalling and repurposes it for activation of viral gene expression. We show that E1A structurally and functionally mimics a cellular A-kinase anchoring protein (AKAP). E1A interacts with and relocalizes protein kinase A (PKA) to the nucleus, likely to virus replication centres, via an interaction with the regulatory subunits of PKA. Binding to PKA requires the N-terminus of E1A, which bears striking similarity to the amphipathic α-helical domain present in cellular AKAPs. E1A also targets the same docking-dimerization domain of PKA normally bound by cellular AKAPs. In addition, the AKAP like motif within E1A could restore PKA interaction to a cellular AKAP in which its normal interaction motif was deleted. During infection, E1A successfully competes with endogenous cellular AKAPs for PKA interaction. E1A's role as a viral AKAP contributes to viral transcription, protein expression and progeny production. These data establish HAdV E1A as the first known viral AKAP. This represents a unique example of viral subversion of a crucial cellular regulatory pathway via structural mimicry of the PKA interaction domain of cellular AKAPs.

Adenovirus E1A recruits the human Paf1 complex to enhance transcriptional elongation.

UNLABELLED: During infection by human adenovirus (HAdV), the proteins encoded by the early region 1A (E1A) gene bind and appropriate components of the cellular transcriptional machinery to activate the transcription of viral early genes. Previously, we identified roles for the human Bre1 (hBre1) and hPaf1 complexes in E1A-mediated transcriptional activation of HAdV early genes. Here we show that E1A binds hBre1 directly and that this complex targets the hPaf1 complex via the Rtf1 subunit. Depletion of hPaf1 reduces E1A-dependent activation of transcription from the E2e, E3, and E4 viral transcription units, and this does not result from a reduced ability of RNA polymerase II to be recruited to the promoter-proximal regions of these genes. In contrast, depletion of hPaf1 reduces the occupancy of RNA polymerase II across these transcription units. This is accompanied by reductions in the level of H3K36 trimethylation, a posttranslational histone modification associated with efficient transcriptional elongation, and the number of full-length transcripts from these genes. Together, these results indicate that E1A uses hBre1 to recruit the hPaf1 complex in order to optimally activate viral early transcription by enhancing transcriptional elongation. IMPORTANCE: This work provides the mechanism by which the hPaf1 complex contributes to E1A-dependent activation of early gene transcription. The work also demonstrates that E1A induces gene expression by stimulating transcriptional elongation, in addition to its better-characterized effects on transcriptional initiation.

Adenovirus E1A targets the DREF nuclear factor to regulate virus gene expression, DNA replication, and growth.

UNLABELLED: The adenovirus E1A gene is the first gene expressed upon viral infection. E1A remodels the cellular environment to maximize permissivity for viral replication. E1A is also the major transactivator of viral early gene expression and a coregulator of a large number of cellular genes. E1A carries out its functions predominantly by binding to cellular regulatory proteins and altering their activities. The unstructured nature of E1A enables it to bind to a large variety of cellular proteins and form new molecular complexes with novel functions. The C terminus of E1A is the least-characterized region of the protein, with few known binding partners. Here we report the identification of cellular factor DREF (ZBED1) as a novel and direct binding partner of E1A. Our studies identify a dual role for DREF in the viral life cycle. DREF contributes to activation of gene expression from all viral promoters early in infection. Unexpectedly, it also functions as a growth restriction factor for adenovirus as knockdown of DREF enhances virus growth and increases viral genome copy number late in the infection. We also identify DREF as a component of viral replication centers. E1A affects the subcellular distribution of DREF within PML bodies and enhances DREF SUMOylation. Our findings identify DREF as a novel E1A C terminus binding partner and provide evidence supporting a role for DREF in viral replication. IMPORTANCE: This work identifies the putative transcription factor DREF as a new target of the E1A oncoproteins of human adenovirus. DREF was found to primarily localize with PML nuclear bodies in uninfected cells and to relocalize into virus replication centers during infection. DREF was also found to be SUMOylated, and this was enhanced in the presence of E1A. Knockdown of DREF reduced the levels of viral transcripts detected at 20 h, but not at 40 h, postinfection, increased overall virus yield, and enhanced viral DNA replication. DREF was also found to localize to viral promoters during infection together with E1A. These results suggest that DREF contributes to activation of viral gene expression. However, like several other PML-associated proteins, DREF also appears to function as a growth restriction factor for adenovirus infection.

The human papillomavirus E7 proteins associate with p190RhoGAP and alter its function.

UNLABELLED: Using mass spectrometry, we identified p190RhoGAP (p190) as a binding partner of human papillomavirus 16 (HPV16) E7. p190 belongs to the GTPase activating protein (GAP) family and is one of the primary GAPs for RhoA. GAPs stimulate the intrinsic GTPase activity of the Rho proteins, leading to Rho inactivation and influencing numerous biological processes. RhoA is one of the best-characterized Rho proteins and is specifically involved in formation of focal adhesions and stress fibers, thereby regulating cell migration and cell spreading. Since this is the first report that E7 associates with p190, we carried out detailed interaction studies. We show that E7 proteins from other HPV types also bind p190. Furthermore, we found that conserved region 3 (CR3) of E7 and the middle domain of p190 are important for this interaction. More specifically, we identified two residues in CR3 of E7 that are necessary for p190 binding and used mutants of E7 with mutations of these residues to determine the biological consequences of the E7-p190 interaction. Our data suggest that the interaction of E7 with p190 dysregulates this GAP and alters the actin cytoskeleton. We also found that this interaction negatively regulates cell spreading on a fibronectin substrate and therefore likely contributes to important aspects of the HPV life cycle or HPV-induced tumorigenesis. IMPORTANCE: This study identifies p190RhoGAP as a novel cellular binding partner for the human papillomavirus (HPV) E7 protein. Our study shows that a large number of different HPV E7 proteins bind p190RhoGAP, and it identifies regions in both E7 and p190RhoGAP which are important for the interaction to occur. This study also highlights the likelihood that the E7-p190RhoGAP interaction may have important biological consequences related to actin organization in the infected cell. These changes could be an important contributor to the viral life cycle and during progression to cancer in HPV-infected cells. Importantly, this work also emphasizes the need for further study in a field which has largely been unexplored as it relates to the HPV life cycle and HPV-induced transformation.

Viral retasking of hBre1/RNF20 to recruit hPaf1 for transcriptional activation.

Upon infection, human adenovirus (HAdV) must activate the expression of its early genes to reprogram the cellular environment to support virus replication. This activation is orchestrated in large part by the first HAdV gene expressed during infection, early region 1A (E1A). E1A binds and appropriates components of the cellular transcriptional machinery to modulate cellular gene transcription and activate viral early genes transcription. Previously, we identified hBre1/RNF20 as a target for E1A. The interaction between E1A and hBre1 antagonizes the innate antiviral response by blocking H2B monoubiquitination, a chromatin modification necessary for the interferon (IFN) response. Here, we describe a second distinct role for the interaction of E1A with hBre1 in transcriptional activation of HAdV early genes. Furthermore, we show that E1A changes the function of hBre1 from a ubiquitin ligase involved in substrate selection to a scaffold which recruits hPaf1 as a means to stimulate transcription and transcription-coupled histone modifications. By using hBre1 to recruit hPaf1, E1A is able to optimally activate viral early transcription and begin the cycle of viral replication. The ability of E1A to target hBre1 to simultaneously repress cellular IFN dependent transcription while activating viral transcription, represents an elegant example of the incredible economy of action accomplished by a viral regulatory protein through a single protein interaction.

The adenovirus 55 residue E1A protein is a transcriptional activator and binds the unliganded thyroid hormone receptor.

The early region 1A (E1A) of human adenovirus types 2 and 5 is differentially spliced to yield five distinct mRNAs that encode different proteins. The smallest E1A RNA transcript encodes a 55 residue (55R) protein that shares only 28 amino acid residues with the other E1A proteins. Even though it is the most abundant E1A transcript at late times post-infection, little is known about the functions of this E1A isoform. In this study, we show that the E1A 55R protein interacts with, and modulates the activity of the unliganded thyroid hormone receptor (TR). We demonstrate that E1A 55R contains a signature motif known as the CoRNR box that confers interaction with the unliganded TR; this motif was originally identified in cellular corepressors. Using a system reconstituted in the yeast Saccharomyces cerevisiae, which lack endogenous TR and TR coregulators, we show that E1A 55R nonetheless differs from cellular corepressors as it functions as a strong co-activator of TR-dependent transcription and that it possesses an intrinsic transcriptional activation domain. These data indicate that the E1A 55R protein functions as a transcriptional regulator.

Vaccinia virus outperforms a panel of other poxviruses as a potent oncolytic agent for the control of head and neck squamous cell carcinoma cell lines.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is the fifth most common cancer worldwide. Existing therapies for advanced tumors have high failure rates and can have severe consequences in terms of pain, disfigurement, and poor speech and swallowing function. New treatment strategies are needed to improve outcomes for patients suffering with this disease and oncolytic viruses represent a promising approach. METHODS: We infected six well-characterized HNSCC cell lines (Cal27, Detroit562, FaDu, SCC4, SCC15, SCC25), with increasing doses of a panel of poxviruses (including myxoma, vaccinia, raccoonpox and tanapox viruses) modified to express green fluorescence protein to determine which virus was the most effective oncolytic agent in cell-based assays. RESULTS: While myxoma, raccoonpox and tanapox displayed differing efficacy in the panel of cell lines, vaccinia virus was the most potent of the tested poxviruses and was highly effective in controlling cell growth in all cell lines. CONCLUSION: Oncolytic poxviruses, particularly vaccinia virus, were effective in killing HNSCC in vitro and hold promise as potential treatments for patients with HNSCC.

The viral etiology of EBV-associated gastric cancers contributes to their unique pathology, clinical outcomes, treatment responses and immune landscape.

Epstein-Barr virus (EBV) is a pathogen known to cause a number of malignancies, often taking years for them to develop after primary infection. EBV-associated gastric cancer (EBVaGC) is one such malignancy, and is an immunologically, molecularly and pathologically distinct entity from EBV-negative gastric cancer (EBVnGC). In comparison with EBVnGCs, EBVaGCs overexpress a number of immune regulatory genes to help form an immunosuppressive tumor microenvironment (TME), have improved prognosis, and overall have an "immune-hot" phenotype. This review provides an overview of the histopathology, clinical features and clinical outcomes of EBVaGCs. We also summarize the differences between the TMEs of EBVaGCs and EBVnGCs, which includes significant differences in cell composition and immune infiltration. A list of available EBVaGC and EBVnGC gene expression datasets and computational tools are also provided within this review. Finally, an overview is provided of the various chemo- and immuno-therapeutics available in treating gastric cancers (GCs), with a focus on EBVaGCs.

Outcomes for potentially Resectable patients undergoing primary chemoradiation treatment for T1-T2 HPV Negative oropharyngeal squamous cell carcinoma.

BACKGROUND: Transoral surgical resectability (TOS) is a prognostic factor for patients with HPV+ T1-2 oropharyngeal squamous cell carcinoma (OPSCC) disease undergoing radiotherapy (RT), but it is unclear whether this holds for HPV-negative (HPV-) patients. We aimed to compare outcomes of potential TOS-candidates vs. non-TOS candidates, among patients who underwent RT/CRT for early T-stage HPV- OPSCC. METHODS: For patients treated with RT/CRT for early T-stage HPV-negative OPSCC between 2014 and 2021, pretreatment imaging was reviewed by four head-and-neck surgeons, masked to clinical outcomes, to assess primary-site suitability for TOS. Extracapsular extension (ECE) was assessed by a head-and-neck neuroradiologist. We compared outcomes based on surgical resectability relating to: (1) the primary site tumor alone, and (2) the primary site plus the absence/presence of ECE (overall assessment). Kaplan-Meier curves for overall survival (OS), disease-specific survival (DSS), and progression-free survival (PFS) were compared using the log-rank test. RESULTS: Seventy patients were included in the analysis. The primary site was TOS-favorable in 46/70 (66%). Based on the overall assessment, 41/70 (58.6%) were TOS-favorable. The 3-year OS, DSS and PFS for primary site TOS-favorable versus unfavorable were OS: 76.9% versus 37.4%; DSS: 78.1% versus 46.2%, PFS: 69.9% versus 41.3%, (log-rank test = 0.01, 0.03, 0.04; respectively). Additionally, patients with an overall assessment of TOS favorability demonstrated better survival outcomes compared with TOS-unfavorable patients (OS: 77.3% vs. 46.2%; DSS: 78.2% vs. 56.5%, PFS: 72.3% vs. 42.1%, log-rank test = 0.01, 0.04, 0.01; respectively). CONCLUSION: Patients with TOS-favorable HPV-negative early T-stage OPSCC have superior survival outcomes than TOS-unfavorable patients.

HPV-negative head and neck cancers with adverse pathological features carry specific molecular changes that are associated with survival.

BACKGROUND: Adverse pathological features following surgery in head and neck squamous cell carcinoma (HNSCC) are strongly associated with survival and guide adjuvant therapy. We investigated molecular changes associated with these features. METHODS: We downloaded data from the Cancer Genome Atlas and Cancer Proteome Atlas HNSCC cohorts. We compared tumors positive versus negative for perineural invasion (PNI), lymphovascular invasion (LVI), extracapsular spread (ECS), and positive margins (PSM), with multivariable analysis. RESULTS: All pathological features were associated with poor survival, as were the following molecular changes: low cyclin E1 (HR = 1.7) and high PKC-alpha (HR = 1.8) in tumors with PNI; six of 13 protein abundance changes with LVI; greater tumor hypoxia and high Raptor (HR = 2.0) and Rictor (HR = 1.6) with ECS; and low p38 (HR = 2.3), high fibronectin (HR = 1.6), low annexin A1 (HR = 3.1), and high caspase-9 (HR = 1.6) abundances with PSM. CONCLUSIONS: Pathological features in HNSCC carry specific molecular changes that may explain their poor prognostic associations.

Metabolic pathway-based subtypes associate glycan biosynthesis and treatment response in head and neck cancer.

Head and Neck Squamous Cell Carcinoma (HNSCC) is a heterogeneous malignancy that remains a significant challenge in clinical management due to frequent treatment failures and pronounced therapy resistance. While metabolic dysregulation appears to be a critical factor in this scenario, comprehensive analyses of the metabolic HNSCC landscape and its impact on clinical outcomes are lacking. This study utilized transcriptomic data from four independent clinical cohorts to investigate metabolic heterogeneity in HNSCC and define metabolic pathway-based subtypes (MPS). In HPV-negative HNSCCs, MPS1 and MPS2 were identified, while MPS3 was enriched in HPV-positive cases. MPS classification was associated with clinical outcome post adjuvant radio(chemo)therapy, with MPS1 consistently exhibiting the highest risk of therapeutic failure. MPS1 was uniquely characterized by upregulation of glycan (particularly chondroitin/dermatan sulfate) metabolism genes. Immunohistochemistry and pilot mass spectrometry imaging analyses confirmed this at metabolite level. The histological context and single-cell RNA sequencing data identified the malignant cells as key contributors. Globally, MPS1 was distinguished by a unique transcriptomic landscape associated with increased disease aggressiveness, featuring motifs related to epithelial-mesenchymal transition, immune signaling, cancer stemness, tumor microenvironment assembly, and oncogenic signaling. This translated into a distinct histological appearance marked by extensive extracellular matrix remodeling, abundant spindle-shaped cancer-associated fibroblasts, and intimately intertwined populations of malignant and stromal cells. Proof-of-concept data from orthotopic xenotransplants replicated the MPS phenotypes on the histological and transcriptome levels. In summary, this study introduces a metabolic pathway-based classification of HNSCC, pinpointing glycan metabolism-enriched MPS1 as the most challenging subgroup that necessitates alternative therapeutic strategies.

Conserved region 3 of human papillomavirus 16 E7 contributes to deregulation of the retinoblastoma tumor suppressor.

The human papillomavirus (HPV) E7 oncoprotein binds cellular factors, preventing or retargeting their function and thereby making the infected cell conducive for viral replication. A key target of E7 is the product of the retinoblastoma susceptibility locus (pRb). This interaction results in the release of E2F transcription factors and drives the host cell into the S phase of the cell cycle. E7 binds pRb via a high-affinity binding site in conserved region 2 (CR2) and also targets a portion of cellular pRb for degradation via the proteasome. Evidence suggests that a secondary binding site exists in CR3, and that this interaction influences pRb deregulation. Additionally, evidence suggests that CR3 also participates in the degradation of pRb. We have systematically analyzed the molecular mechanisms by which CR3 contributes to deregulation of the pRb pathway by utilizing a comprehensive series of mutations in residues predicted to be exposed on the surface of HPV16 E7 CR3. Despite differences in the ability to interact with cullin 2, all CR3 mutants degrade pRb comparably to wild-type E7. We identified two specific patches of residues on the surface of CR3 that contribute to pRb binding independently of the high-affinity CR2 binding site. Mutants within CR3 that affect pRb binding are less effective than the wild-type E7 in overcoming pRb-induced cell cycle arrest. This demonstrates that the interaction between HPV16 E7 CR3 and pRb is functionally important for alteration of the cell cycle.

Cellular GCN5 is a novel regulator of human adenovirus E1A-conserved region 3 transactivation.

The largest isoform of adenovirus early region 1A (E1A) contains a unique region termed conserved region 3 (CR3). This region activates viral gene expression by recruiting cellular transcription machinery to the early viral promoters. Recent studies have suggested that there is an optimal level of E1A-dependent transactivation required by human adenovirus (hAd) during infection and that this may be achieved via functional cross talk between the N termini of E1A and CR3. The N terminus of E1A binds GCN5, a cellular lysine acetyltransferase (KAT). We have identified a second independent interaction of E1A with GCN5 that is mediated by CR3, which requires residues 178 to 188 in hAd5 E1A. GCN5 was recruited to the viral genome during infection in an E1A-dependent manner, and this required both GCN5 interaction sites on E1A. Ectopic expression of GCN5 repressed transactivation by both E1A CR3 and full-length E1A. In contrast, RNA interference (RNAi) depletion of GCN5 or treatment with the KAT inhibitor cyclopentylidene-[4-(4'-chlorophenyl)thiazol-2-yl]hydrazone (CPTH2) resulted in increased E1A CR3 transactivation. Moreover, activation of the adenovirus E4 promoter by E1A was increased during infection of homozygous GCN5 KAT-defective (hat/hat) mouse embryonic fibroblasts (MEFs) compared to wild-type control MEFs. Enhanced histone H3 K9/K14 acetylation at the viral E4 promoter required the newly identified binding site for GCN5 within CR3 and correlated with repression and reduced occupancy by phosphorylated RNA polymerase II. Treatment with CPTH2 during infection also reduced virus yield. These data identify GCN5 as a new negative regulator of transactivation by E1A and suggest that its KAT activity is required for optimal virus replication.