Abstract 2389: The role of pyroptosis in the tumor microenvironment of head and neck squamous cell carcinoma (HNSCC)

Abstract Background: HNSCC exhibits diverse tumor microenvironments (TME), influencing progression and therapeutic responses. Pyroptosis, a form of inflammatory programmed cell death may be associated with TME’s and anti-tumor immunity. This study investigates pyroptosis-related gene expression in HNSCC TMEs, classified into immune-enriched (IE), immune-enriched fibrotic (IE/F), immune-depleted (ID), and fibrotic (F). Methods: RNA-seq data from TCGA HNSCC cohort were stratified into two TME subtypes. Tumors were grouped into IE (IE & IE/F) and ID (ID & F) for comparative analysis. Pyroptosis-related genes were curated from 838 gene sets using the MSigDB and validated through STRING protein-protein interaction analysis. Pathway enrichment was evaluated via supervised and unsupervised analyses using Gene Set Enrichment Analysis (GSEA) and Gene Set Variation Analysis (GSVA). Key contributors were identified through leading-edge analysis, with survival associations assessed using Cox proportional hazards modeling. Preliminary analysis using EcoTyper and CIBERSORTx has identified cell states and cancer ecotypes associated with TME. Results: Unsupervised GSEA identified 31 enriched pathways in HPV-negative samples (FDR q-value <0.05), including Regulation of Cell Killing, Interferon Responsive Genes, and Pyroptosis. Leading-edge analysis revealed overlapping immune-regulatory pathways, such as Positive Regulation of Lymphocyte Apoptotic Process and Role of Phospholipids in Phagocytosis, with shared signals from genes including CD2, PDCD1, and TIGIT. Reactome Pyroptosis (FDR=2.04e-25) highlighted molecular interactions involving GSDME and CASP3. Core genes in IE phenotypes included CD3E, IL12RB1, and FASLG. Supervised analysis of pyroptosis pathways (NES=1.71, FDR q-value=0.069) identified 35 enriched genes such as CASP1, NLRC4, and GSDMD. STRING analysis highlighted pathways including cytotoxic T-cell pyroptosis (FDR=1.03e-03) and immune-associated pyroptosis (FDR=1.2e-14). GSVA revealed 424 significant pathway differences between immune-enriched and immune-depleted phenotypes, including Pyroptosis (p=0.0004) and Cell Killing (p=0.0021). IE phenotypes upregulated pathways including Interferon Gamma Signaling (p=0.0008) and Positive Regulation of Leukocyte Activation (p=0.0013). Cox analysis linked 82 pathways including Reactome Mitophagy (HR 3.56, p=3.13e-05) and Reactome Autophagy (HR 5.69, p=0.0003) to improved OS and PFS. Conclusions: Pyroptosis and immune-regulatory pathways are more strongly associated with IE TME phenotypes and correlate with improved OS and PFS. These findings support pyroptosis as a modulator of prognosis in HNSCC, which might inform therapeutic mechanisms in overcoming treatment-resistant disease. Future study will apply this analysis to clinical trial RNA-seq data, among patients who underwent anti-PD-1 therapy. Citation Format: Hani Samarah, Julian Jackson, Hannah L. Kenny, Zachary Kaplan, Parvesh Kumar, Zhao Lin, Adam Luginbuhl, Eric Mastrolonardo, Alban Linnenbach, Kelly Bridgham, Teresa Alnemri, Joseph Curry. The role of pyroptosis in the tumor microenvironment of head and neck squamous cell carcinoma (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 2389.