Abstract

After nearly a decade of stagnation, the standard of care for head and neck squamous cell carcinoma (HNSCC) has expanded into the immunotherapy field with the incorporation of immune checkpoint blockade into perioperative anti-PD-1 therapy for many patients with resectable, locally advanced disease. Despite dramatic and curative results in some patients, most tumors fail to respond, and some patients may even be harmed through delay of surgical intervention or paradoxical accelerated tumor growth under therapy—an increasingly documented phenomenon known as “hyperprogression”. These clinical challenges reflect a central gap in HNSCC biology: the mechanisms that determine whether anti-PD-1 therapy reinvigorates productive anti-tumor immunity or instead fails to control, or even drives acceleration of, disease remain incompletely understood. This dissertation attempts to address that gap by defining malignant cell-intrinsic programs that mark or even drive response and resistance to immune checkpoint blockade in HNSCC. Chapter 1 establishes the critical concepts for this work by reviewing the epidemiology, clinical burden, and therapeutic landscape of HNSCC, with emphasis on the emergence of immune checkpoint blockade and the limitations of current biomarkers such as PD-L1 combined positive score. This chapter also introduces tumor epigenetics as a mechanism of therapeutic resistance, focusing on how malignant cells may adopt dynamic, non-genetic transcriptional states in response to environmental pressures arising from therapy. These concepts provide the foundation for studying how interferon signaling, inflammatory memory, and epithelial- mesenchymal plasticity shape the tumor immune microenvironment. Chapter 2 investigates mechanisms of response to anti-PD-1 therapy. Using single-cell RNA sequencing of matched pre-treatment and post-treatment HNSCC tumors from patients treated with neoadjuvant pembrolizumab, this chapter identifies a malignant-cell program characterized by interferon-response genes and major histocompatibility complex class II expression. Functional studies in syngeneic murine models demonstrate that malignant-cell MHC-II expression is dependent on IFN-γ signaling and is associated with effective anti-tumor immunity, increased T cell infiltration, and reduced myeloid predominance. However, genetic loss of malignant-cell MHC-II itself does not abrogate therapeutic response, indicating that (in this model) MHC-II functions primarily as a marker of a broader IFN-γ-driven malignant-cell state rather than as functional antigen presentation driving therapeutic response. These findings establish malignant-cell-intrinsic IFN-γ signaling as a key feature of productive PD-1 blockade in HNSCC. Chapter 3 examines how the same IFN-γ signaling pathway that marks response can be subverted by malignant cells into a mechanism of resistance. This chapter demonstrates that chronic interferon exposure induces an epithelial alarmin-like transcriptional program, termed Epi-alarmin, in malignant cells. After interferon withdrawal, a subset of Epi-alarmin cytokines remains persistently expressed, forming a durable cytokine memory state. This inflammatory epigenetic memory recruits immunosuppressive neutrophils, excludes cytotoxic T cells, and confers resistance to immune checkpoint blockade. Mechanistically, the establishment and maintenance of this state require the epithelial ETS-family transcription factor Elf3, which binds cytokine loci and preserves chromatin accessibility at neutrophil-recruiting genes. Loss of Elf3 disrupts the Epi-alarmin program, restores a more T cell-inflamed tumor microenvironment, and improves response to anti-PD-1 therapy. These findings reveal that interferon signaling has context-dependent effects: acute IFN-γ signaling reflects productive anti-tumor immunity, whereas chronic interferon exposure can be stably integrated into malignant cell chromatin, thus driving immune suppression and therapeutic resistance. Finally, Chapter 4 defines a second malignant-cell resistance program centered on hybrid epithelial-mesenchymal cell states. This chapter shows that hybrid EM programs in HNSCC are supported by BRD4-driven super-enhancer activity, and that pharmacologic or genetic disruption of BRD4 suppresses hybrid EM gene expression and invasive phenotypes. Single-cell profiling of murine oral cancer models reveals a continuum of epithelial, hybrid EM, and mesenchymal states in vivo, with aggressive tumors enriched for intermediate and mesenchymal programs. Similar to the tumor-specific MHC-II and Epi-alarmin programs, these states are not purely cell- intrinsic. Instead, they are shaped by fibroblast-derived signals, most notably TGF-β. Finally, hybrid EM gene expression is associated with resistance to immune checkpoint blockade, linking tumor cell plasticity, invasion, stromal interaction, and immune evasion. Together, these studies argue that response and resistance to immunotherapy in HNSCC are governed not only by immune-cell abundance or PD-L1 expression, but also by more biologically nuanced malignant-cell states. Clinically, this work provides evidence for moving beyond PD-L1 combined positive score (CPS) toward biomarkers that capture the functional state of the tumor-immune interface. Malignant-cell IFN/MHC-II expression may identify tumors undergoing productive immune engagement, whereas ELF3-driven inflammatory memory and hybrid EM programs may indicate tumors at risk for resistance or progression under checkpoint blockade. Deeper identification and refinement of these programs in human tumors may ultimately enable more precise selection of patients likely to benefit from anti-PD-1 therapy, while sparing non-responders from ineffective treatment—instead affording them access to alternative therapeutic strategies with greater potential for benefit.

Committee Chair

Sidharth Puram

Committee Members

Robi Mitra, Charles Kaufman; Michael Meers; Nathan Singh

Degree

Doctor of Philosophy (PhD)

Author's Department

Biology & Biomedical Sciences (Molecular Genetics & Genomics)

Author's School

Graduate School of Arts and Sciences

Document Type

Dissertation

Date of Award

7-8-2026

Language

English (en)

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Available for download on Wednesday, July 07, 2027

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Biology Commons

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