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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.

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

The Hippo pathway is frequently dysregulated in cancer, leading to the unrestrained activity of its downstream targets, YAP/TAZ, and aberrant tumor growth. However, the precise mechanisms leading to YAP/TAZ activation in most cancers is still poorly understood. Analysis of large tissue collections revealed YAP activation in most head and neck squamous cell carcinoma (HNSCC), but only 29.8% of HNSCC cases present genetic alterations in the FAT1 tumor suppressor gene that may underlie persistent YAP signaling. EGFR is overexpressed in HNSCC and many other cancers, but whether EGFR controls YAP activation is still poorly understood. Here, we discover that EGFR activates YAP/TAZ in HNSCC cells, but independently of its typical signaling targets, including PI3K. Mechanistically, we find that EGFR promotes the phosphorylation of MOB1, a core Hippo pathway component, and the inactivation of LATS1/2 independently of MST1/2. Transcriptomic analysis reveals that erlotinib, a clinical EGFR inhibitor, inactivates YAP/TAZ. Remarkably, loss of LATS1/2, resulting in aberrant YAP/TAZ activity, confers erlotinib resistance on HNSCC and lung cancer cells. Our findings suggest that EGFR-YAP/TAZ signaling plays a growth-promoting role in cancers harboring EGFR alterations, and that inhibition of YAP/TAZ in combination with EGFR might be beneficial to prevent treatment resistance and cancer recurrence.

The dominant paradigm for HPV carcinogenesis includes integration into the host genome followed by expression of E6 and E7 (E6/E7). We explored an alternative carcinogenic pathway characterized by episomal E2, E4, and E5 (E2/E4/E5) expression. Half of HPV positive cervical and pharyngeal cancers comprised a subtype with increase in expression of E2/E4/E5, as well as association with lack of integration into the host genome. Models of the E2/E4/E5 carcinogenesis show p53 dependent enhanced proliferation in vitro, as well as increased susceptibility to induction of cancer in vivo. Whole genomic expression analysis of the E2/E4/E5 pharyngeal cancer subtype is defined by activation of the fibroblast growth factor receptor (FGFR) pathway and this subtype is susceptible to combination FGFR and mTOR inhibition, with implications for targeted therapy.

Tipifarnib is a potent and highly selective inhibitor of farnesyltransferase (FTase). FTase catalyzes the posttranslational attachment of farnesyl groups to signaling proteins that are required for localization to cell membranes. Although all RAS isoforms are FTase substrates, only HRAS is exclusively dependent upon farnesylation, raising the possibility that HRAS-mutant tumors might be susceptible to tipifarnib-mediated inhibition of FTase. Here, we report the characterization of tipifarnib activity in a wide panel of HRAS-mutant and wild-type head and neck squamous cell carcinoma (HNSCC) xenograft models. Tipifarnib treatment displaced both mutant and wild-type HRAS from membranes but only inhibited proliferation, survival, and spheroid formation of HRAS-mutant cells. In vivo, tipifarnib treatment induced tumor stasis or regression in all six HRAS-mutant xenografts tested but displayed no activity in six HRAS wild-type patient-derived xenograft (PDX) models. Mechanistically, drug treatment resulted in the reduction of MAPK pathway signaling, inhibition of proliferation, induction of apoptosis, and robust abrogation of neovascularization, apparently via effects on both tumor cells and endothelial cells. Bioinformatics and quantitative image analysis further revealed that FTase inhibition induces progressive squamous cell differentiation in tipifarnib-treated HNSCC PDXs. These preclinical findings support that HRAS represents a druggable oncogene in HNSCC through FTase inhibition by tipifarnib, thereby identifying a precision therapeutic option for HNSCCs harboring HRAS mutations.

Patients with advanced cancers are treated with combined radiotherapy and chemotherapy, however curability is poor and treatment side effects severe. Drugs sensitizing tumors to radiotherapy have been developed to improve cell kill, but tumor specificity remains challenging. To achieve tumor selectivity of small molecule radiosensitizers, we tested as a strategy active tumor targeting using peptide-based drug conjugates. We attached an inhibitor of the DNA damage response to antibody or cell penetrating peptides. Antibody drug conjugates honed in on tumor overexpressed cell surface receptors with high specificity but lacked efficacy when conjugated to the DNA damage checkpoint kinase inhibitor AZD7762. As an alternative approach, we synthesized activatable cell penetrating peptide scaffolds that accumulated within tumors based on matrix metalloproteinase cleavage. While matrix metalloproteinases are integral to tumor progression, they have proven therapeutically elusive. We harnessed these pro-tumorigenic extracellular proteases to spatially guide radiosensitizer drug delivery using cleavable activatable cell penetrating peptides. Here, we tested the potential of these two drug delivery platforms targeting distinct tumor compartments in combination with radiotherapy and demonstrate the advantages of protease triggered cell penetrating peptide scaffolds over antibody drug conjugates to deliver small molecule amine radiosensitizers.

Prostate cancer (PCa) innervation contributes to the progression of PCa. However, the precise impact of innervation on PCa cells is still poorly understood. By focusing on muscarinic receptors, which are activated by the nerve-derived neurotransmitter acetylcholine, we show that muscarinic receptors 1 and 3 (m1 and m3) are highly expressed in PCa clinical specimens compared with all other cancer types, and that amplification or gain of their corresponding encoding genes (CHRM1 and CHRM3, respectively) represent a worse prognostic factor for PCa progression free survival. Moreover, m1 and m3 gene gain or amplification is frequent in castration-resistant PCa (CRPC) compared with hormone-sensitive PCa (HSPC) specimens. This was reflected in HSPC-derived cells, which show aberrantly high expression of m1 and m3 under androgen deprivation mimicking castration and androgen receptor inhibition. We also show that pharmacological activation of m1 and m3 signaling is sufficient to induce the castration-resistant growth of PCa cells. Mechanistically, we found that m1 and m3 stimulation induces YAP activation through FAK, whose encoding gene, PTK2 is frequently amplified in CRPC cases. Pharmacological inhibition of FAK and knockdown of YAP abolished m1 and m3-induced castration-resistant growth of PCa cells. Our findings provide novel therapeutic opportunities for muscarinic-signal-driven CRPC progression by targeting the FAK-YAP signaling axis.

Gastrointestinal stromal tumor (GIST), the most common sarcoma, is characterized by KIT protein overexpression, and tumors are frequently driven by oncogenic KIT mutations. Targeted inhibition of KIT revolutionized GIST therapy and ushered in the era of precision medicine for the treatment of solid malignancies. Here, we present the first use of a KIT-specific DNA aptamer for targeted labeling of GIST. We found that an anti-KIT DNA aptamer bound cells in a KIT-dependent manner and was highly specific for GIST cell labeling in vitro Functionally, the KIT aptamer bound extracellular KIT in a manner similar to KIT mAb staining, and was trafficked intracellularly in vitro The KIT aptamer bound dissociated primary human GIST cells in a mutation agnostic manner such that tumors with KIT and PDGFRA mutations were labeled. In addition, the KIT aptamer specifically labeled intact human GIST tissue ex vivo, as well as peritoneal xenografts in mice with high sensitivity. These results represent the first use of an aptamer-based method for targeted detection of GIST in vitro and in vivo.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Tobacco use is the main risk factor for HNSCC, and tobacco-associated HNSCCs have poor prognosis and response to available treatments. Recently approved anti-PD-1 immune checkpoint inhibitors showed limited activity (≤20%) in HNSCC, highlighting the need to identify new therapeutic options. For this, mouse models that accurately mimic the complexity of the HNSCC mutational landscape and tumor immune environment are urgently needed. Here, we report a mouse HNSCC model system that recapitulates the human tobacco-related HNSCC mutanome, in which tumors grow when implanted in the tongue of immunocompetent mice. These HNSCC lesions have similar immune infiltration and response rates to anti-PD-1 (≤20%) immunotherapy as human HNSCCs. Remarkably, we find that >70% of HNSCC lesions respond to intratumoral anti-CTLA-4. This syngeneic HNSCC mouse model provides a platform to accelerate the development of immunotherapeutic options for HNSCC.

Aberrant activation of the PI3K-mTOR signaling pathway occurs in >80% of head and neck squamous cell carcinomas (HNSCC), and overreliance on this signaling circuit may in turn represent a cancer-specific vulnerability that can be exploited therapeutically. mTOR inhibitors (mTORi) promote tumor regression in genetically defined and chemically induced HNSCC animal models, and encouraging results have been recently reported. However, the mTOR-regulated targets contributing to the clinical response have not yet been identified. Here, we focused on EIF4E-BP1 (4E-BP1), a direct target of mTOR that serves as key effector for protein synthesis. A systematic analysis of genomic alterations in the PIK3CA-mTOR pathway in HNSCC revealed that 4E-BP1 is rarely mutated, but at least one 4E-BP1 gene copy is lost in over 35% of the patients with HNSCC, correlating with decreased 4E-BP1 protein expression. 4E-BP1 gene copy number loss correlated with poor disease-free and overall survival. Aligned with a tumor-suppressive role, 4e-bp1/2 knockout mice formed larger and more lesions in models of HNSCC carcinogenesis. mTORi treatment or conditional expression of a mutant 4E-BP1 that cannot be phosphorylated by mTOR was sufficient to disrupt the translation-initiation complex and prevent tumor growth. Furthermore, CRISPR/Cas9-targeted 4E-BP1 HNSCC cells resulted in reduced sensitivity to mTORi in vitro and in vivo. Overall, these findings indicate that in HNSCC, mTOR persistently restrains 4E-BP1 via phosphorylation and that mTORi can restore the tumor-suppressive function of 4E-BP1. Our findings also support 4E-BP1 expression and phosphorylation status as a mechanistic biomarker of mTORi sensitivity in patients with HNSCC. SIGNIFICANCE: These findings suggest that EIF4E-BP1 acts as a tumor suppressor in HNSCC and that 4E-BP1 dephosphorylation mediates the therapeutic response to mTORi, providing a mechanistic biomarker for future precision oncology trials.