Inactivating NOTCH1 mutations in head and neck squamous cell carcinoma (HNSCC) were described over a decade ago, suggesting a tumor suppressor function - unlike its oncogenic role in other tumors. Today, much debate persists regarding a putative oncogenic role in HNSCC as well, with reports that NOTCH1 signaling drives tumor growth and a cancer stem cell (CSC) phenotype. In this work, comprehensive experiments unequivocally demonstrate that NOTCH1 is a tumor suppressor in HNSCC regardless of mutation or activation status and that it reduces CSC frequency. We developed a signature of NOTCH1 activation showing the pathway is associated with very early differentiation, an altered tumor microenvironment, and better prognosis. Clarifying whether NOTCH1 occasionally functions as an oncogenic driver in HNSCC is crucial to prognosis and personalized therapy. The results presented unify the field, reconcile conflicting data, and provide critical insights into the biological and clinical significance of NOTCH1, with broader implications in other squamous carcinomas with NOTCH1 mutations.

OBJECTIVES:Oral cavity head and neck squamous cell carcinoma (HNSCC) represents a major global health burden, with over 40,000 new cases annually in the United States and >500,000 worldwide. Despite advances in surgery, radiation therapy, chemotherapy, and immunotherapy, overall survival rates have remained largely stagnant over the past three decades, and over half of patients ultimately die from the disease. Although immune checkpoint inhibitors (ICIs) have improved outcomes for a subset of patients with recurrent or metastatic HNSCC, the majority gain limited benefit. Thus, novel therapeutic strategies that enhance responses to immunotherapy are urgently needed. The ATR-CHK1 signaling pathway has recently been implicated in immune modulation, suggesting that ATR inhibition may potentiate antitumor immunity. This study investigates the therapeutic efficacy of the novel ATR inhibitor AZD6738, alone and in combination with anti-PD-1 therapy, in a syngeneic mouse model of HNSCC. MATERIALS AND METHODS:An orthotopic syngeneic mouse model was used to assess in vivo treatment efficacy. Immune profiling of tumors following AZD6738 treatment was performed by immunohistochemistry and flow cytometry. Conditioned medium from cocultures of tumor and bone marrow cells treated with AZD6738 was analyzed by flow cytometry to evaluate immune cell populations. Additional coculture experiments with bone marrow-derived cells and splenic T cells assessed T cell activation and proliferation. In vitro assays, including clonogenic survival, western blotting, and ATR shRNA knockdown, were used to confirm drug specificity and explore molecular mechanisms. RNA sequencing of tumor samples was conducted to evaluate cytokine expression and immune-related gene signatures. RESULTS:AZD6738 significantly enhanced the efficacy of anti-PD-1 therapy by remodeling the tumor microenvironment. Treatment increased cytotoxic T cell infiltration and activity while reducing regulatory T cells and immunosuppressive myeloid cells. ATR inhibition promoted M1 macrophage polarization and modulated cytokines supporting T cell activation. Mechanistically, enhanced immune responses were associated with DNA damage-induced activation of the cGAS/STING pathway and reduced STAT3 phosphorylation. Transcriptomic analyses revealed that combination therapy upregulated interferon signaling and suppressed epithelial-mesenchymal transition pathways in the MOC1 tumor model. CONCLUSION:AZD6738 enhances antitumor immunity through cytotoxic T cells and innate immune mechanisms, supporting its further development in combination with anti-PD-1 therapy for HNSCC.

Background. Hyperactivation of NRF2 is an established factor contributing to chemoradiation resistance in head and neck squamous cell carcinoma (HNSCC). In tumors with KEAP1 loss or mutations in the oncogenic PIK3CA pathway, NRF2 accumulates, enhancing antioxidant defenses that mitigate the cytotoxic effects of cisplatin and ionizing radiation. An alternative approach to directly targeting NRF2 exploits the PI3K/AKT/GSK3 axis, which regulates a KEAP1-independent pathway for NRF2 proteasomal degradation. Methods. We evaluated the effects of the dual PI3K/mTOR inhibitor gedatolisib on NRF2-signaling and phenotype in various human and murine HNSCC cell lines, as well as genetically engineered mouse models (GEMMs) with conditional Keap1 deletion and orthotopic xenografts of the cisplatin-resistant HN30R8 line. Proliferative responses were tracked using real-time IncuCyte imaging and label-free holotomographic microscopy. NRF2 protein regulation was assessed via western blot, nuclear/cytoplasmic fractionation, and RT-qPCR. The role of GSK3 and the proteasome in the gedatolisib-induced reduction of NRF2 was confirmed through pharmacological rescue with GSK3 and proteasome inhibitors and further validated by siRNA knockdown of GSK3-α, GSK3-β, β-TrCP, and SPOP. Results. Gedatolisib inhibited AKT phosphorylation and decreased NRF2 protein levels in a dose- and time-dependent manner across all tested cell lines, including those derived from GEMMs with Keap1 deletion. This reduction was post-translational, as NFE2L2 mRNA was upregulated via FoxO transcription factor activation, while NRF2 protein was directed towards GSK3-dependent, β-TrCP/SPOP-mediated proteasomal degradation. Holotomographic live-cell imaging revealed that cells treated with gedatolisib primarily underwent necrotic cell death, which was accompanied by mitochondrial accumulation and growth arrest. Gedatolisib effectively suppressed tumor growth in both Keap1 wild-type and Keap1 -deleted GEMMs. In orthotopic HN30R8 cisplatin-resistant xenografts, gedatolisib was the primary driver of antitumor activity. Additionally, the combination of gedatolisib and radiation showed synergistic tumor suppression according to the multiplicative model in a replicated experiment. Conclusions. Gedatolisib effectively overcomes NRF2-driven resistance in HNSCC by engaging a degradation pathway that does not rely on KEAP1. The drug simultaneously activated NRF2 transcription through FoxO and accelerated the degradation of NRF2 protein via the GSK3/β-TrCP pathway, resulting in decreased NRF2 protein levels. These findings support the potential of gedatolisib as a chemoradiosensitizer in HNSCC clinical trials.

Abstract Background Cisplatin remains the standard systemic therapy for the definitive treatment of head and neck squamous cell carcinoma (HNSCC), however, resistance to cisplatin continues to be a major barrier to effective treatment, particularly in tumors with NRF2 hyperactivation. Recent studies identify secreted phosphoprotein 1 (SPP1/osteopontin) as a key NRF2 target frequently overexpressed in cancers, where it drives aggressive tumor behavior, metastasis, chemoresistance, and, in some cases, immune suppression. Our recent data highlight SPP1 as one of the top 10 NRF2-upregulated genes in cisplatin-resistant HNSCC. However, its specific role in therapy resistance and metastasis in HNSCC remains unclear. Here, we investigate whether targeting SPP1 can suppress tumor aggressiveness and improve cisplatin response in HNSCC. Methods Using established human HNSCC cell lines and mouse models, we utilized conventional western blotting, cell invasion, functional proteomics and high resolution spatial transcriptomics to examine the role of SPP1 in driving tumor progression and metastasis in therapy-resistant HNSCC. Results Targeted suppression of SPP1 improved cisplatin sensitivity, inhibited tumor invasion and metastasis both in vitro and in vivo and reduced expression of several metastatic signaling proteins in NRF2-hyperactivated HNSCC. Proteomic analysis revealed that silencing SPP1 led to dysregulation of critical oncogenic and metastatic signaling pathways, including MAPK, AKT/mTOR, FAK, and PAK1. Spatial transcriptomic analysis uncovered a potential mechanistic interaction between SPP1, integrins and CD44 receptors in both primary and metastatic HNSCC. Spatial annotation and enrichment analyses using HALLMARK revealed gene set signatures of interferon and EMT present in cell clusters with SPP1 expression in both the primary tumor and lung metastases. Finally, increased expression of SPP1 was found to be poor prognostic factor and significantly correlated with NFE2L2/KEAP1 mutational status and higher tumor grade in HNSCC patients. Conclusions Targeting dysregulated SPP1 improved cisplatin sensitivity and suppressed tumor invasion and metastasis in NRF2-hyperactivated HNSCC, underscoring the therapeutic potential of SPP1 inhibitors to improve patient outcomes.

Supplementary Table S4A. The KNC genes enriched in cluster 1 in HNSCC cells; Supplementary Table S4B. Gene activity score of cell surface markers in cluster 1 in HNSCC cells.

Whole exome sequencing showing mutational status in the parental HN30-P versus HN30-R8 cisplatin resistant cell lines.

Supplementary figures with figure legends

Gene list for mouse leukocyte subsets

Supplementary Table S5A. Bulk-level single cell ATAC sequencing analysis showing immediate (short-term) chromatin openings (peaks) in HN30-P cells following CDDP treatment; Supplementary Table S5B. Bulk-level single cell ATAC sequencing analysis showing long-term chromatin openings (peaks) gained in cisplatin-resistant HN30-R8 cells following CDDP treatment.

Supplementary Materials and Methods

Cisplatin resistant HNSCC cells migrate faster than their Cisplatin sensitive counterparts.

NRF2 regulated genes significantly upregulated in distant metastasis compared to paired primary tumors in HN30-R8 CDDP group.

Keap1 tumor growth influenced by fractionated radiation therapy

The status and co-occurrence of mutations in patients with HNSCC

About 5% of cancers are caused by human papillomavirus (HPV), most of which are resistant to immune checkpoint inhibitors (ICIs). Alisertib (Ali), an Aurora kinase A inhibitor, may sensitize these cancers to ICIs by inducing apoptosis and DNA damage. This study investigates combining Ali with ICIs to enhance antitumor immunity and overcome resistance. We assessed Ali’s effects on apoptosis, DNA damage, and immunogenic cell death in HPV-positive cancer cell lines. Ali was tested alone or with ICIs in the mEER model, a surrogate for HPV+ oropharyngeal cancers. Changes in the tumor immune microenvironment (TIME) were analyzed by flow cytometry. RNA-seq evaluated gene expression across four groups: control, Ali, anti-CTLA-4, and combination. Gene set enrichment and Gene Ontology (GO) analyses identified key biological processes.Combination therapy with Ali and anti-CTLA-4 significantly reduced tumor size and improved survival, with 75% of treated mice surviving at 60 days compared to 0% of controls. Ali induced increased cleaved caspase-3 and PARP expression in tumor tissues, indicating enhanced cancer cell apoptosis. Flow cytometry showed enhanced T-cell polyfunctionality, with increased IFN-γ and granzyme production in tumor-infiltrating lymphocytes, along with reduced T-cell exhaustion (e.g., LAG3). A significant reduction in the Gr-1low Ly6G+ granulocytic subset of myeloid-derived suppressor cells (MDSCs) suggested an improved immune microenvironment. RNA-seq identified 22, 059 protein-coding genes with ≥1.5-fold increase compared to controls. Of these, 400 genes were upregulated by Ali, 3 by anti-CTLA-4, and 640 by the combination, with 191 genes upregulated in both Ali and combination groups. GO analysis revealed that shared genes activated immune-related processes like cell surface receptor signaling, macrophage activation, and T-cell cytokine regulation. In the Ali group, immune activation was linked to cytokine production and immune effector responses, while the combination showed enrichment for metabolic processes, suggesting metabolic reprogramming as a mechanism for enhanced immune responses and efficacy. The combination therapy increased CD8+ central memory T-cells, CD4+ memory T-cells, and elevated naive CD4+ T-cells, indicating enhanced T-cell activation and recruitment to the tumor, correlating with increased immune cell infiltration and improved survival. The combination of Ali and anti-CTLA-4 enhances outcomes in an HPV-driven murine model by boosting immune activation and reshaping the TIME. RNA-seq analysis shows that Ali drives immune responses, further amplified by anti-CTLA-4, promoting both immune and metabolic reprogramming. These findings suggest that combining Ali with ICI could overcome resistance and improve therapeutic outcomes in HPV-positive cancers. Soma Ghosh, Madison P. O’Hara, Tuhina Mazumdar, Lacin Yapindi, Li Shen, Jing Wang, Mitchell J. Frederick, Jagannadha K. Sastry, Faye M. Johnson. Aurora-A inhibition sensitizes HPV-driven cancers to anti-CTLA-4 immunotherapy by modulating the tumor immune microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3094.

GO and KEGG pathway analysis of top KEAP1/NRF2/CUL3 (axis regulated genes and their association with clinical outcome in HNSCC.

Restoration of wildtype KEAP1 improves survival in mice.

Nrf2 knockdown in Keap1-deficient murine HNSCC cell lines and RT responses

Relationship between TP53 status and the NRF2/KEAP1/CUL3 pathway in OCSCC TCGA samples.