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BackgroundNatural killer (NK) cells are key effector cells of antitumor immunity. However, tumors can acquire resistance programs to escape NK cell-mediated immunosurveillance. Identifying mechanisms that mediate this resistance enables us to define approaches to improve immune-mediate antitumor activity. In previous studies from our group, a genome-wide CRISPR-Cas9 screen identified Charged Multivesicular Body Protein 2A (CHMP2A) as a novel mechanism that mediates tumor intrinsic resistance to NK cell activity.MethodsHere, we use an immunocompetent mouse model to demonstrate that CHMP2A serves as a targetable regulator of not only NK cell-mediated immunity but also other immune cell populations. Using the recently characterized murine 4MOSC model system, a syngeneic, tobacco-signature murine head and neck squamous cell carcinoma model, we deleted mCHMP2A using CRISPR/Cas9-mediated knock-out (KO), following orthotopic transplantation into immunocompetent hosts.ResultsWe found that mCHMP2A KO in 4MOSC1 cells leads to more potent NK-mediated tumor cell killing in vitro in these tumor cells. Moreover, following orthotopic transplantation, KO of mCHMP2A in 4MOSC1 cells, but not the more immune-resistant 4MOSC2 cells enables both T cells and NK cells to better mediate antitumor activity compared with wild type (WT) tumors. However, there was no difference in tumor development between WT and mCHMP2A KO 4MOSC1 or 4MOSC2 tumors when implanted in immunodeficient mice. Mechanistically, we find that mCHMP2A KO 4MOSC1 tumors transplanted into the immunocompetent mice had significantly increased CD4+T cells, CD8+T cells. NK cell, as well as fewer myeloid-derived suppressor cells (MDSC).ConclusionsTogether, these studies demonstrate that CHMP2A is a targetable inhibitor of cellular antitumor immunity.

Uveal melanoma (UM) is the most prevalent cancer of the eye in adults, driven by activating mutation of GNAQ/GNA11; however, there are limited therapies against UM and metastatic UM (mUM). Here, we perform a high-throughput chemogenetic drug screen in GNAQ-mutant UM contrasted with BRAF-mutant cutaneous melanoma, defining the druggable landscape of these distinct melanoma subtypes. Across all compounds, darovasertib demonstrates the highest preferential activity against UM. Our investigation reveals that darovasertib potently inhibits PKC as well as PKN/PRK, an AGC kinase family that is part of the "dark kinome." We find that downstream of the Gαq-RhoA signaling axis, PKN converges with ROCK to control FAK, a mediator of non-canonical Gαq-driven signaling. Strikingly, darovasertib synergizes with FAK inhibitors to halt UM growth and promote cytotoxic cell death in vitro and in preclinical metastatic mouse models, thus exposing a signaling vulnerability that can be exploited as a multimodal precision therapy against mUM.

OBJECTIVES: This study aimed to evaluate the outcomes of a hands-on simulation-based course with emphasis on procedural techniques, clinical reasoning, and communication skills developed to improve junior Otolaryngology - Head and Neck Surgery (OHNS) residents preparedness in managing otolaryngologic emergencies. METHODS: Junior OHNS residents and faculty from residency programs in California, Nevada, and Arizona participated in this workshop in 2020 and 2021. The stations featured airway management techniques, ultrasound-guided needle aspiration, nasoseptal hematoma evacuation, and facial fracture repair using various models and cadavers. Participants completed a pre-workshop survey, post-workshop survey, and 2-month follow-up survey that assessed resident anxiety and confidence in three OHNS emergency situations across knowledge, manual skills, and teamwork using a 5-point Likert scale. RESULTS: Pre-workshop surveys reported the least anxiety and most confidence in teamwork, but the most anxiety and least confidence in technical skills and knowledge related to foreign body retrieval and airway management. Immediately post-workshop participants reported significant reductions in anxiety and increases in confidence, largest in the manual skills domain, in foreign body retrieval (anxiety: -0.99, confidence: +0.95, p

Immune checkpoint blockade (ICB) targeting PD-1 and CTLA-4 has revolutionized cancer treatment. However, many cancers do not respond to ICB, prompting the search for additional strategies to achieve durable responses. G-protein-coupled receptors (GPCRs) are the most intensively studied drug targets but are underexplored in immuno-oncology. Here, we cross-integrated large singe-cell RNA-sequencing datasets from CD8+ T cells covering 19 distinct cancer types and identified an enrichment of Gαs-coupled GPCRs on exhausted CD8+ T cells. These include EP2, EP4, A2AR, β1AR and β2AR, all of which promote T cell dysfunction. We also developed transgenic mice expressing a chemogenetic CD8-restricted Gαs-DREADD to activate CD8-restricted Gαs signaling and show that a Gαs-PKA signaling axis promotes CD8+ T cell dysfunction and immunotherapy failure. These data indicate that Gαs-GPCRs are druggable immune checkpoints that might be targeted to enhance the response to ICB immunotherapies.

AbstractWork over the past several decades has identified that aberrations in the ErbB signaling pathways are key drivers of oncogenesis, and concurrent efforts to discover targetable vulnerabilities to counter this aberrant oncogenic signaling offer tremendous promise in treating a host of human cancers. These efforts have been centered primarily on EGFR (also known as HER1), leading to the discovery of the first targeted therapies approved for head and neck cancer. More recently, HER2 and HER3 signaling pathways have been identified as highly dysregulated in head and neck cancer. This review highlights the HER2 and HER3 signaling pathways and clinical efforts to target these receptors and their aberrant signaling to treat head and neck squamous cell carcinomas and other head and neck malignancies, including salivary gland carcinomas. This includes the use of small molecule inhibitors and blocking antibodies, both as single agents or as part of multimodal precision targeted and immunotherapies.

Despite the promise of immune checkpoint inhibition (ICI), therapeutic responses remain limited. This raises the possibility that standard of care treatments delivered in concert may compromise the tumor response. To address this, we employ tobacco-signature head and neck squamous cell carcinoma murine models in which we map tumor-draining lymphatics and develop models for regional lymphablation with surgery or radiation. We find that lymphablation eliminates the tumor ICI response, worsening overall survival and repolarizing the tumor- and peripheral-immune compartments. Mechanistically, within tumor-draining lymphatics, we observe an upregulation of conventional type I dendritic cells and type I interferon signaling and show that both are necessary for the ICI response and lost with lymphablation. Ultimately, we provide a mechanistic understanding of how standard oncologic therapies targeting regional lymphatics impact the tumor response to immune-oncology therapy in order to define rational, lymphatic-preserving treatment sequences that mobilize systemic antitumor immunity, achieve optimal tumor responses, control regional metastatic disease, and confer durable antitumor immunity.

Head and neck squamous cell carcinoma (HNSCC) ranks sixth in cancer incidence worldwide and has a 5-year survival rate of only 63%. Immunotherapies-principally immune checkpoint inhibitors (ICI), such as anti-PD-1 and anti-CTLA-4 antibodies that restore endogenous antitumor T-cell immunity-offer the greatest promise for HNSCC treatment. Anti-PD-1 has been recently approved for first-line treatment of recurrent and metastatic HNSCC; however, less than 20% of patients show clinical benefit and durable responses. In addition, the clinical application of ICI has been limited by immune-related adverse events (irAE) consequent to compromised peripheral immune tolerance. Although irAEs are often reversible, they can become severe, prompting premature therapy termination or becoming life threatening. To address the irAEs inherent to systemic ICI therapy, we developed a novel, local delivery strategy based upon an array of soluble microneedles (MN). Using our recently reported syngeneic, tobacco-signature murine HNSCC model, we found that both systemic and local-MN anti-CTLA-4 therapy lead to >90% tumor response, which is dependent on CD8 T cells and conventional dendritic cell type 1 (cDC1). However, local-MN delivery limited the distribution of anti-CTLA-4 antibody from areas distal to draining lymphatic basins. Employing Foxp3-GFPDTR transgenic mice to interrogate irAEs in vivo, we found that local-MN delivery of anti-CTLA-4 protects animals from irAEs observed with systemic therapy. Taken together, our findings support the exploration of MN-intratumoral ICI delivery as a viable strategy for HNSCC treatment with reduced irAEs, and the opportunity to target cDC1s as part of multimodal treatment options to boost ICI therapy.

PurposeUveal melanoma is the most common eye cancer in adults. Approximately 50% of patients with uveal melanoma develop metastatic uveal melanoma (mUM) in the liver, even after successful treatment of the primary lesions. mUM is refractory to current chemo- and immune-therapies, and most mUM patients die within a year. Uveal melanoma is characterized by gain-of-function mutations in GNAQ/GNA11, encoding Gαq proteins. We have recently shown that the Gαq-oncogenic signaling circuitry involves a noncanonical pathway distinct from the classical activation of PLCβ and MEK-ERK. GNAQ promotes the activation of YAP1, a key oncogenic driver, through focal adhesion kinase (FAK), thereby identifying FAK as a druggable signaling hub downstream from GNAQ. However, targeted therapies often activate compensatory resistance mechanisms leading to cancer relapse and treatment failure.Experimental designWe performed a kinome-wide CRISPR-Cas9 sgRNA screen to identify synthetic lethal gene interactions that can be exploited therapeutically. Candidate adaptive resistance mechanisms were investigated by cotargeting strategies in uveal melanoma and mUM in vitro and in vivo experimental systems.ResultssgRNAs targeting the PKC and MEK-ERK signaling pathways were significantly depleted after FAK inhibition, with ERK activation representing a predominant resistance mechanism. Pharmacologic inhibition of MEK and FAK showed remarkable synergistic growth-inhibitory effects in uveal melanoma cells and exerted cytotoxic effects, leading to tumor collapse in uveal melanoma xenograft and liver mUM models in vivo.ConclusionsCoupling the unique genetic landscape of uveal melanoma with the power of unbiased genetic screens, our studies reveal that FAK and MEK-ERK cotargeting may provide a new network-based precision therapeutic strategy for mUM treatment.See related commentary by Harbour, p. 2967.

Immune checkpoint blockade (ICB) therapy has revolutionized head and neck squamous cell carcinoma (HNSCC) treatment, but

Recent advancements in the development of immunotherapies have raised the hope for patients with locally-advanced HNSCC (LA-HNSCC) to achieve improved oncologic outcomes without the heavy burden of treatment-related morbidity. While there are several ongoing late phase clinical trials that seek to determine whether immunotherapy can be effectively employed in the definitive setting, initial results from concurrent immuno-radiotherapy therapy trials have not shown strong evidence of benefit. Encouragingly, evidence from preclinical studies and early-phase neoadjuvant studies have begun to show potential pathways forward, with therapeutic combinations and sequences that intentionally spare tumor draining lymphatics in order to maximize the synergy between definitive local therapy and immunotherapy. The intent of this review is to summarize the scientific rationale and current clinical evidence for employing immunotherapy for LA-HNSCC as well as the ongoing efforts and challenges to determine how to optimally deliver and sequence immunotherapy alongside traditional therapeutics. In both the preclinical and clinical settings, we will discuss the application of immunotherapies to both surgical and radiotherapeutic management of HNSCC.

Innate immune cells quickly infiltrate the site of pathogen entry and not only stave off infection but also initiate antigen presentation and promote adaptive immunity. The recruitment of innate leukocytes has been well studied in the context of extracellular bacterial and fungal infection but less during viral infections. We have recently shown that the understudied cytokine Interleukin (IL)-17D can mediate neutrophil, natural killer (NK) cell and monocyte infiltration in sterile inflammation and cancer. Herein, we show that early immune cell accumulation at the peritoneal site of infection by mouse cytomegalovirus (MCMV) is mediated by IL-17D. Mice deficient in IL-17D or the transcription factor Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), an inducer of IL-17D, featured an early decreased number of innate immune cells at the point of viral entry and were more susceptible to MCMV infection. Interestingly, we were able to artificially induce innate leukocyte infiltration by applying the Nrf2 activator tert-butylhydroquinone (tBHQ), which rendered mice less susceptible to MCMV infection. Our results implicate the Nrf2/IL-17D axis as a sensor of viral infection and suggest therapeutic benefit in boosting this pathway to promote innate antiviral responses.

Cells undergoing xenobiotic or oxidative stress activate the transcription factor nuclear factor erythroid-derived 2-like 2 (Nrf2), which initiates an intrinsic "stress surveillance" pathway. We recently found that the cytokine IL-17D effects a form of extrinsic stress surveillance by inducing antitumor immunity, but how IL-17D is regulated remains unknown. Here, we show that Nrf2 induced IL-17D in cancer cell lines. Moreover, both Nrf2 and IL-17D were induced in primary tumors as well as during viral infection in vivo. Expression of IL-17D in tumors and virally infected cells is essential for optimal protection of the host as il17d(-/-) mice experienced a higher incidence of tumors and exacerbated viral infections compared to wild-type (WT) animals. Moreover, activating Nrf2 to induce IL-17D in established tumors led to natural killer cell-dependent tumor regression. These data demonstrate that Nrf2 can initiate both intrinsic and extrinsic stress surveillance pathways and highlight the use of Nrf2 agonists as immune therapies for cancer and infection.

The process of cancer immunoediting generates a repertoire of cancer cells that can persist in immune-competent hosts. In its most complex form, this process begins with the elimination of highly immunogenic unedited tumor cells followed by the escape of less immunogenic edited cells. Although edited tumors can release immunosuppressive factors, it is unknown whether unedited tumors produce cytokines that enhance antitumor function. Utilizing gene microarray analysis, we found the cytokine interleukin 17D (IL-17D) was highly expressed in certain unedited tumors but not in edited mouse tumor cell lines. Moreover, forced expression of IL-17D in edited tumor cells induced rejection by stimulating MCP-1 production from tumor endothelial cells, leading to the recruitment of natural killer (NK) cells. NK cells promoted M1 macrophage development and adaptive immune responses. IL-17D expression was also decreased in certain high-grade and metastatic human tumors, suggesting that it can be targeted for tumor immune therapy.

Cancer immunoediting is the process whereby immune cells protect against cancer formation by sculpting the immunogenicity of developing tumors. Although the full process depends on innate and adaptive immunity, it remains unclear whether innate immunity alone is capable of immunoediting. To determine whether the innate immune system can edit tumor cells in the absence of adaptive immunity, we compared the incidence and immunogenicity of 3'methylcholanthrene-induced sarcomas in syngeneic wild-type, RAG2(-/-), and RAG2(-/-)x γc(-/-) mice. We found that innate immune cells could manifest cancer immunoediting activity in the absence of adaptive immunity. This activity required natural killer (NK) cells and interferon γ (IFN-γ), which mediated the induction of M1 macrophages. M1 macrophages could be elicited by administration of CD40 agonists, thereby restoring editing activity in RAG2(-/-)x γc(-/-) mice. Our results suggest that in the absence of adaptive immunity, NK cell production of IFN-γ induces M1 macrophages, which act as important effectors during cancer immunoediting.

Cancer immunoediting is the process whereby immune cells protect against cancer formation by sculpting the immunogenicity of developing tumors. Although the full process depends on innate and adaptive immunity, it remains unclear whether innate immunity alone is capable of immunoediting. To determine whether the innate immune system can edit tumor cells in the absence of adaptive immunity, we compared the incidence and immunogenicity of 3'methylcholanthrene-induced sarcomas in syngeneic wild-type, RAG2(-/-), and RAG2(-/-)x γc(-/-) mice. We found that innate immune cells could manifest cancer immunoediting activity in the absence of adaptive immunity. This activity required natural killer (NK) cells and interferon γ (IFN-γ), which mediated the induction of M1 macrophages. M1 macrophages could be elicited by administration of CD40 agonists, thereby restoring editing activity in RAG2(-/-)x γc(-/-) mice. Our results suggest that in the absence of adaptive immunity, NK cell production of IFN-γ induces M1 macrophages, which act as important effectors during cancer immunoediting.

Primary central nervous system (CNS) tumors cause significant morbidity and mortality in both adults and children. While some of the genetic and molecular mechanisms of neuro-oncogenesis are known, much less is known about possible epigenetic contributions to disease pathophysiology. Over the last several decades, chronic viral infections have been associated with a number of human malignancies. In primary CNS malignancies, two families of viruses, namely polyomavirus and herpesvirus, have been detected with varied frequencies in a number of pediatric and adult histological tumor subtypes. However, establishing a link between chronic viral infection and primary CNS malignancy has been an area of considerable controversy, due in part to variations in detection frequencies and methodologies used among researchers. Since a latent viral neurotropism can be seen with a variety of viruses and a widespread seropositivity exists among the population, it has been difficult to establish an association between viral infection and CNS malignancy based on epidemiology alone. While direct evidence of a role of viruses in neuro-oncogenesis in humans is lacking, a more plausible hypothesis of neuro-oncomodulation has been proposed. The overall goals of this review are to summarize the many human investigations that have studied viral infection in primary CNS tumors, discuss potential neuro-oncomodulatory mechanisms of viral-associated CNS disease and propose future research directions to establish a more firm association between chronic viral infections and primary CNS malignancies.