Abstract Loss of p53 function is a major driver of poor outcomes in HNSCC, with over 70% of tumors harboring deactivating TP53 mutations. Despite decades of effort, mutant p53 remains “undruggable”. Cholesterol is essential for membrane integrity and oncogenic signaling, and elevated cholesterol levels correlate with aggressiveness across many cancers. In HNSCC, patients whose tumors express high levels of cholesterol-upregulating genes have a 50% higher risk of death, highlighting this pathway as a clinically relevant therapeutic target. We previously showed that HPV+ HNSCC cells retain wild-type (WT) p53 activity and that p53 transcriptionally regulates multiple metabolic pathways, including cholesterol-regulating genes. These observations led us to investigate how p53 loss rewires cholesterol metabolism and whether this metabolic shift creates a targetable vulnerability in p53-deficient HNSCC. Silencing WT p53 in HPV+ HNSCC cells (siRNA or CRISPRi) increased intracellular cholesterol and proliferation - an effect reversed by cholesterol deprivation. This p53 deficiency also elevated expression and maturation of SREBP1/2, master transcriptional regulators of cholesterol metabolism, along with multiple downstream SREBP targets. Elevated cholesterol and activated SREBP1/2 were similarly observed in HPV-negative p53-mutant HNSCC lines, indicating that p53-dependent cholesterol metabolism is a broader function independent of HPV status. In HNSCC patient-derived xenografts (PDXs), p53-mutant tumors exhibited higher expression and preferential nuclear localization of SREBP1 compared to p53-WT tumors, confirming cholesterol pathway reprogramming in vivo. To test pathway dependency, we examined the effects of SREBP1 genetic ablation (via siRNA or in CRISPR knock-out datasets) in HNSCC and other squamous carcinoma lines. SREBP1 loss selectively reduced viability in p53-deficient cells compared with WT counterparts. Pharmacologic inhibition of SREBP1/2 with the small molecule fatostatin also produced an average 2.7-fold greater cytotoxic response in p53-deficient versus p53-proficient HNSCC lines. In isogenic cell line-derived xenograft (CDX) models, fatostatin treatment (315 mg/kg over 32 days) significantly suppressed growth of p53-deficient tumors. A pan-cancer pharmacogenomic screen of >900 cell lines further identified p53 deficiency as a determinant of fatostatin sensitivity. Together, these findings demonstrate that p53 loss confers dependency on SREBP-driven cholesterol metabolism and support therapeutic targeting of this pathway in a genomically defined subset of HNSCC. Citation Format: Jovanka Gencel-Augusto, Nuo Tian, Akshat Singhal, Hua Li, Liam Woerner, Arthur Goldberg, Daniel E. Johnson, Jennifer Rubin Grandis. Targeting cholesterol pathways in p53 deficient head and neck cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2999.

The mitogen-activated protein kinase kinase (MEK)/extracellular-regulated kinase (ERK) signaling pathway is hyperactivated in myeloid leukemias characterized by differentiation blockade, suggesting a potential role in disruption of myeloid differentiation. However, other reports indicate that MEK/ERK activation is required for normal myeloid differentiation. To investigate the in vivo role of MEK/ERK hyperactivation in myeloid differentiation and leukemia development we generated transgenic mice with doxycycline (DOX)-inducible expression of constitutively active MEK1 (CA-MEK1) under the control of the myeloid-selective MRP8 promoter. Two independent transgenic lines (lines A and B) were generated. Strikingly, both lines developed epithelial abnormalities following DOX induction of CA-MEK1. Line A mice exhibited pervasive skin/epithelial thickening, while line B mice developed papillomas. Prominent induction of CA-MEK1 was detected in epidermis, but not dermis, and was accompanied by epithelial cell hyperplasia. Line B mice also exhibited CA-MEK1 induction and ERK1/2 phosphorylation/activation in bone marrow and blood. Induction of CA-MEK1 over a range of 8-33 weeks, however, failed to alter the frequency of monocytes and granulocytes in the blood of line B mice. Collectively, our findings demonstrate that the MRP8 promoter is active in both myeloid and epithelial tissues. Moreover, we confirm earlier reports that hyperactivation of MEK/ERK signaling in epithelial tissues promotes epithelial hyperplasia. In addition, our findings indicate that hyperactivation of the MEK/ERK pathway alone is insufficient to alter myeloid differentiation and initiate leukemia development. We propose that MEK/ERK hyperactivation likely acts to promote proliferation or cell survival in leukemias where differentiation has been blocked by other genetic or epigenetic events.

Non-steroidal anti-inflammatory drugs (NSAIDs), primarily aspirin, have long been studied for their anti-cancer properties. Epidemiologic studies have reported diminished rates of several cancers in regular NSAID users. NSAIDs inhibit cyclooxygenase (COX) enzymes, thus decreasing the production of pro-inflammatory prostaglandins, which are known to stimulate tumor growth. Efforts to characterize the molecular features of human cancer, such as The Cancer Genome Atlas (TCGA), have revealed the presence of genetic alterations that activate the Phosphoinositide 3-kinase (PI3K) signaling pathway in a large subset of cancers, including head and neck squamous cell carcinoma (HNSCC). Mutation or amplification of the PIK3CA oncogene, or decreased expression of the tumor suppressor protein phosphatase and tensin homolog (PTEN), is found in one-third of HNSCCs. Cumulative evidence from retrospective clinical analyses and preclinical laboratory studies has demonstrated the anti-tumor effects of NSAIDs, including aspirin, in PI3K-altered HNSCC. In a recent trial, daily aspirin significantly decreased disease recurrence in PI3K-altered colorectal cancer, thus providing a compelling justification for a prospective trial of aspirin in HNSCC. Given the prevalence of PI3K alterations in HNSCC, aspirin has potential to benefit nearly 1 million patients with HNSCC annually worldwide.

PIK3CA encodes the catalytic subunit of phosphoinositide 3-kinase (PI3K) enzyme and is the most commonly mutated oncogene in head and neck squamous cell carcinoma (HNSCC). This study aimed to identify potential therapeutic targets in HNSCC harboring mutant PIK3CA. We used CRISPR interference (CRISPRi)-based genome-wide screening methodology to reveal targetable genetic dependencies in PIK3CA-mutated HNSCC. Screening was conducted in an HPV-positive HNSCC cell line, UM-SCC-47, engineered to express the canonical E545K PIK3CA mutant. We identified 34 genes co-dependent on PIK3CA E545K mutation, including 5 genes in the neddylation pathway (NEDD8, NEDD8-MDP-1 and NAE1, USP8, UBA3). Validation experiments confirmed the essential role of NEDD8, NEDD8-MDP-1, and NAE1, indicating a novel regulatory mechanism in PIK3CA E545K-mutated HNSCC. Our findings suggest that PIK3CA mutation may serve as a predictive biomarker for neddylation inhibitor therapy in a subpopulation of HNSCC.

STAT3 is an oncogenic transcription factor that activates cancer cell signaling and induces an immunosuppressive immune environment, making it an attractive therapeutic target. Transcription factors are exceptionally challenging targets and there are no Food and Drug Administration-approved STAT3 inhibitors. We previously reported positive pharmacodynamics of a linear STAT3 decoy oligonucleotide administered intratumorally in a phase 0 trial in patients with head and neck cancer squamous cell carcinoma (HNSCC). Here, we describe the anti-tumor and immune effects of a systemically administered cyclic STAT3 decoy (CS3D) in immunocompetent HNSCC murine models and the safety and efficacy of CS3D in a clinical trial in pet cats with HNSCC. Responders in the clinical trial (35% disease control rate) showed significant differences in selected peripheral blood immune parameters as well as elevated PD-1 expression in the tumors compared with non-responders. These findings support a clinical trial of CS3D in HNSCC patients.

Head and neck squamous cell carcinoma (HNSCC) is a major global health challenge. Inactivation of the tumor suppressor p53 is the most frequent driver event in this malignancy. p53 inactivation occurs either through TP53 mutations in human papilloma virus (HPV)-negative cases or via HPV-mediated p53 degradation in HPV-positive (HPV+) cases, where most tumors retain a wild-type (WT) TP53 allele. This underscores the critical role ofp53-regulated processes in HNSCC pathogenesis. Clinically, HPV+ HNSCC has significantly better outcomes than HPV-negative disease. However, approximately 10% of HPV+ HNSCC tumors harbor TP53 mutations, suggesting selective pressure to suppress p53 signaling beyond viral degradation. In this study, we demonstrate that HPV+ TP53-WTHNSCC cells have residual, tumor suppressive p53 activity. Clinically, patients with HPV+ TP53-WT tumors exhibit significantly better survival than those with HPV+ TP53-mutantor HPV-negative tumors. In vitro, WT p53 loss in HPV+ HNSCC cells enhances proliferation, migration, and invasion, and transcriptomic analysis confirms ongoing p53-dependent gene regulation. HPV+ TP53-WT tumors also display tumor-suppressive gene methylation patterns, fewer chromosomal alterations, and reduced PI3K-AKT signaling compared to TP53-mutant HPV+ cases. Notably, p53 loss increases expression of the PI3K catalytic subunit p110α, suppresses the PI3K-AKT inhibitor INPP5D, and sensitizes cells to pharmacologic PI3Kinhibition. Together, our findings challenge the prevailing view that p53 is completely inactivated in HPV+ HNSCC and reveal tumor suppressive, p53-driven mechanisms that persist in these tumors. These insights highlight a potential role for TP53-based stratification in guiding treatment decisions and suggest new therapeutic vulnerabilities in HPV+ HNSCC.

Purpose: Heterozygous mutations in the gene encoding the caspase-8 protease are present in approximately 10% of head and neck squamous cell carcinoma (HNSCC) tumors, where studies indicate they block apoptosis induced by death ligands. However, the role of caspase-8 mutations in HNSCC development in vivo and their influence on response to programmed cell death-1 (PD-1) targeting agents remain unclear. Experimental Design: To investigate the role of caspase-8 mutation in HNSCC, we generated mice with an epithelium-specific, heterozygous knock-in of a representative HNSCC-associated caspase-8 mutation (D305G), regulated by the endogenous caspase-8 promoter. The impact of this mutation was assessed following carcinogen (4-nitroquinoline-1-oxide, 4NQO) treatment in drinking water. Additionally, to evaluate the effects of heterozygous caspase-8 mutations on anti-PD-1 responsiveness, murine HNSCC tumors were established in immunocompetent mice using MOC1 cells engineered to express mutant caspase-8.Results: Mice harboring the D305G caspase-8 mutation developed a higher number of tongue tumors per mouse and showed a greater incidence of advanced invasive carcinomas following 4NQO treatment compared to wild-type caspase-8 controls. In MOC1 tumor models, knockout or heterozygous expression of HNSCC-associated caspase-8 mutants significantly reduced sensitivity to anti-PD-1 therapy, in contrast to tumors derived from parental MOC1 cells. Conclusions: These findings reveal that caspase-8 mutations enhance carcinogen-induced HNSCC progression and confer resistance to anti-PD-1 therapy. Further investigation into caspase-8 mutations as biomarkers for poor immunotherapy response in HNSCC patients is warranted. Citation Format: Zhibin Cui, Liam C Woerner, Steven R Long, Christopher N Peterson, Nathan K VanLandingham, Andrew Nazarenko, Jennifer R Grandis, Daniel E Johnson. Caspase-8 mutation promotes head and neck squamous cell carcinoma development and resistance to anti-PD-1 [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Functional and Genomic Precision Medicine in Cancer: Different Perspectives, Common Goals; 2025 Mar 11-13; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(5 Suppl):Abstract nr B009.

Genetic alterations in genes encoding components of the PI3K/AKT/mTOR signaling pathway are frequently observed in head and neck squamous cell carcinoma (HNSCC), breast cancer, and a variety of other human malignancies. In particular, PIK3CA, encoding the p110α catalytic subunit of PI3K enzyme, is altered in approximately 30 % of HNSCC tumors and 37 % of breast cancer tumors. In addition, loss of PTEN protein, a negative regulator of PI3K signaling, occurs in roughly one-third of HNSCC. Here, we review the impact of these alterations on the growth and metabolism of cancer cells and summarize progress that has been made in the development and clinical evaluation of inhibitors that directly target p110α and related proteins. We also describe emerging approaches that are identifying unique vulnerabilities and targeting opportunities in tumors characterized by PIK3CA or PTEN alterations.

Head and neck squamous cell carcinoma (HNSCC) is a major global health challenge. Inactivation of the tumor suppressor p53 is the most frequent molecular event in this malignancy. p53 inactivation occurs either through TP53 mutations in HPV-negative cases or via HPV-mediated p53 degradation in HPV-positive (HPV+) cases, where most retain a wild-type (WT) TP53 allele. This underscores the critical role of p53-regulated processes in HNSCC pathogenesis. Clinically, HPV+ HNSCC is associated with significantly better outcomes than HPV-negative cases. Despite HPV E6-mediated degradation of p53, 10% of HPV+ HNSCC cases have mutant TP53 alleles, suggesting an additional need to suppress p53 signaling. In this study, we demonstrate that HPV+ TP53-WT HNSCC cells have residual p53 activity, with tumor-suppressive effects. Specifically, ablation of p53 in HPV+ TP53-WT HNSCC cells lead to increased proliferation, migration, and invasion. RNA-sequencing revealed that p53 regulates gene transcription (both activation and repression) in the presence of HPV, and we confirmed that these functions are p53-specific. Human tumor data further reveal that among HPV+ HNSCC cases, those with WT TP53 have significantly better survival outcomes than HPV+ cases with TP53 mutations. Notably, HPV+ TP53-WT HNSCC tumors show decreased PI3K-AKT signaling, increased gene methylation in oncogenic pathways, and fewer chromosomal alterations compared to HPV+ TP53-mutant tumors. These findings challenge the prevailing notion that p53 is entirely inactivated in HPV+ HNSCC and provide critical insights into HNSCC-specific, p53-driven tumor suppression mechanisms with implications for targeted therapies in HNSCC. ### Competing Interest Statement The authors have declared no competing interest.

Pyroptosis is an inflammatory programmed cell death recently identified as a crucial cellular process in various diseases, including cancers. Unlike other forms of cell death, canonical pyroptosis involves the specific cleavage of gasdermin by caspase-1, resulting in cell membrane damage and the release of the pro-inflammatory cytokines IL-1β and IL-18. Initially observed in innate immune cells responding to external pathogens or internal death signals, pyroptotic cell death has now been observed in numerous cell types. Recent studies have extensively explored different ways to trigger pyroptotic cell death in solid tumors, presenting a promising avenue for cancer treatment. This review outlines the mechanisms of both canonical and noncanonical pyroptosis pertinent to cancer and primarily focuses on various biomolecules that can induce pyroptosis in malignancies. This strategy aims not only to eliminate cancer cells but also to promote an improved tumor immune microenvironment. Furthermore, emerging research indicates that targeting pyroptotic pathways may improve the effectiveness of existing cancer treatments, making them more potent against resistant tumor types, offering new hope for overcoming treatment resistance in aggressive malignancies.

Lymphopenia is associated with poor outcomes and is exacerbated by conventional chemoradiation therapies in head and neck squamous cell carcinoma (HNSCC). NT-I7 (rhIL-7-hyFc, efineptakin alfa; NeoImmuneTech, Inc.), a long-acting human IL-7, has the potential to restore lymphocyte populations, modulate immune responses, and enhance anti-tumor activity. This study investigated NT-I7’s effects on lymphocyte populations and its synergy with cisplatin and immune checkpoint inhibitors (ICIs) in syngeneic, immunocompetent HNSCC mouse models. The impact of NT-I7 was studied in two syngeneic tumor models, MOC1 and MOC22. Flow cytometry and Cytometry by Time of Flight (CyTOF) analyses were used to evaluate NT-I7 effects on peripheral and intratumoral immune cells. Anti-tumor efficacy was assessed following NT-I7 treatment alone, in combination with cisplatin, or with anti-PD-1 therapy. Mice bearing syngeneic MOC1 and MOC22 tumors exhibited significant lymphopenia, characterized by reductions in CD4+ and CD8+ T-cells, Tregs, NK cells, and NKT cells, resembling clinical observations in HNSCC patients. NT-I7 treatment significantly increased lymphocyte levels in both spleen and tumor, with notable induction of CD8+ T-cells and NKT cells, peaking at day 7 and day 4 post-treatment, respectively, and minimal impact on intratumoral Treg levels. NT-I7 modestly inhibited MOC1 tumor growth and potently suppressed MOC22 tumor growth. Combining NT-I7 with cisplatin significantly enhanced anti-tumor activity in MOC1 tumors, accompanied by increased numbers of peripheral and intratumoral CD4+, CD8+, and NKT cells. Furthermore, NT-I7 augmented the efficacy of anti-PD-1 therapy in MOC1 tumors, resulting in greater tumor growth inhibition compared to either treatment alone. CyTOF analysis revealed preferential expansion of CD8+ Tpex cells, a key subset of tumor-specific cells that expand in response to ICIs, highlighting NT-I7’s role in overcoming lymphopenia and potentiating anti-tumor immunity. NT-I7 effectively increases lymphocyte populations in HNSCC preclinical models that demonstrate tumor-associated lymphopenia, and enhances the efficacy of cisplatin and anti-PD-1 in HNSCC preclinical tumor models. Zhibin Cui, Jacqueline L. Yee, Hua Li, Yan Zeng, Liam Woerner, Alexandra A. Wolfarth, Sara Ferrando-Martinez, Hyunseok Kang, Jennifer R. Grandis, Matthew H. Spitzer, Daniel E. Johnson. NT-I7 increases lymphocytes and enhances chemotherapy and immunotherapy efficacy in HNSCC [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 2152.

Mutations in the Fanconi Anemia (FA) pathway lead to a rare genetic disease that increases risk of bone marrow failure, acute myeloid leukemia, and solid tumors. FA patients have a 500 to 800-fold increase in head and neck squamous cell carcinoma compared to the general population and the treatments for these malignancies are ineffective and limited due to the deficiency in DNA damage repair. Using unbiased CRISPR-interference screening, we found the loss of FA pathway function renders cells dependent on key exocytosis genes such as SNAP23. Further investigation revealed that loss of FA pathway function induced deficiencies in lysosomal health, dysregulation of autophagy and increased lysosomal exocytosis. The compromised cellular state caused by the loss of FA genes is accompanied by decreased lysosome abundance and increased lysosomal membrane permeabilization in cells. We found these signatures in vitro across multiple cell types and cell lines and in clinically relevant FA patient cancers. Our findings are the first to connect the FA pathway to lysosomal exocytosis and thus expands our understanding of FA as a disease and of induced dependencies in FA mutant cancers.

Fanconi anemia (FA) confers a high risk (~700-fold increase) of solid tumor formation, most often head and neck squamous cell carcinoma (HNSCC). FA germline DNA repair defects preclude administration of most chemotherapies, and prior hematopoietic stem cell transplantation limits the use of immunotherapy. Thus, surgery and judicious delivery of radiation offer the only treatment options, with most patients dying from their cancers. A paucity of preclinical models has limited the development of new treatments. Here, we report what to our knowledge are the first patient-derived xenografts (PDXs) of FA-associated HNSCC (FA-HNSCC) and highlight the efficacy of FDA-approved EGFR-targeted therapies in tumors with high EGFR and phosphorylated EGFR levels and the activity of the FDA-approved B-cell lymphoma 2 (Bcl-2) inhibitor venetoclax in a FA-HNSCC PDX overexpressing Bcl-2. These findings support the development of precision medicine approaches for FA-HNSCC.

Increased PI3K signaling as a result of PIK3CA mutation or amplification or decreased expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is one of the most common alterations in head and neck squamous cell carcinoma (HNSCC). PTEN negatively regulates PI3K signaling and its downstream effectors including COX2. COX2 mediates the synthesis of prostaglandin E2 (PGE2) which contributes to immunosuppression in the tumor microenvironment. PGE2 also binds to one or more EP receptors (EP1-EP4) and promotes the growth of tumor cells via activation of EP2 and EP4. However, the role of PGE2 in PTEN-deficient HNSCC is incompletely understood. In this study, we assessed PGE2 signaling in PTEN-deficient HNSCC and evaluated the effect of aspirin or TPST-1495, a dual EP2/EP4 antagonist, on the growth of PTEN knockout and PIK3CA-altered HNSCC tumors in immunocompetent mice. Our results demonstrated that aspirin selectively inhibits the growth of PTEN knockout HNSCC tumors. TPST-1495 inhibited tumor growth and substantially increased the antitumor activity of the immune checkpoint inhibitor anti-PD1. To date, there are no FDA-approved therapies for PI3K pathway-altered HNSCC. Our findings suggest that NSAIDs demonstrate antitumor activity in PTEN-deficient or PI3K-altered tumors whereas EP2/EP4 targeting may augment FDA-approved anti-PD1 therapy in HNSCC.

Background: Human papilloma virus (HPV)-associated head and neck squamous cell carcinoma (HNSCC) is an emerging epidemic and a subset of HPV-positive patients experience aggressive disease with metastases. The CYLD gene is frequently altered in HPV-positive HNSCC, but the role of these alterations in disease progression is poorly understood. Methods: We identified 11 HPV-positive HNSCC patients with CYLD alterations and assessed their clinical course. We also characterized a unique, HPV-positive, metastatic, HNSCC patient-derived xenograft (PDX). Results: All 11 patients developed metastasis with reduced overall survival when compared with metastatic HPV-positive patients with wild-type CYLD. The metastatic PDX harbored a CYLD mutation (S371*) and exhibited reduced expression of connexin 43, a potentially antimetastatic protein. We also investigated the functional impact of the S371* mutation, as well as 2 CYLD mutations from our 11-patient cohort. Conclusion: Our findings indicate that alterations in CYLD in HPV-positive HNSCC are associated with metastasis and poor prognosis.

AREAS COVERED:Here we describe novel agents, their mechanism(s) of action, preclinical results, and ongoing clinical trials in HNSCC. EXPERT OPINION:Established therapeutic targets in HNSCC include EGFR (cetuximab) and PD-1 (pembrolizumab and nivolumab). Despite the detection of many other possible targets in HNSCC cell lines and patient tumors, no other therapies have successfully advanced to date. Identification of predictive biomarkers may guide the use of targeted agents and combination therapies. Clinical trials supported by strong preclinical data in relevant models are more likely to advance treatment options.

Clinical characteristics of patients prior to surgery and PDX establishment

<p>Blockade of the PI3K pathway with BKM120 or BEZ235 increases HER3/AKT signaling</p>

PDF file - 122K, STAT3 decoy treatment does not inhibit cell viability or STAT3 target gene expression in A4 STAT3 null cells

<p>Pan-phospho-RTK profiling identifies HER3 to be hyper-phosphorylated after BYL719 treatment in HPV(+) cell lines</p>