Date of Award

January 2019

Document Type

Open Access Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

Wendell G. Yarbrough, MD, MMHC, FACS

Second Advisor

Natalia Issaeva, PhD

Abstract

The incidence of HPV-positive head and neck squamous cell carcinoma (HNSCC) con-tinues to rise. Though HPV positivity is correlated with improved survival, up to a quar-ter of these tumors recur or metastasize despite aggressive therapy. Currently, there areno biomarkers that can identify HPV-positive HNSCC patients who would benefit fromreduced doses of radiation–which when given at full dose carries significant morbidity.Through analysis of a limited cohort of The Cancer Genome Atlas (TCGA), we previ-ously showed that two genes, TRAF3 and CYLD, were frequently deleted or mutated inHPV-positive HNSCC. TRAF3 and CYLD are functionally related negative regulatorsof the transcription factor NF-κB. In HNSCC, TRAF3 and CYLD mutations were cor-related with increased NF-κB activity, the maintenance of HPV episomes, and improvedpatient survival. Thus, we hypothesize that a subset of HPV-positive HNSCC arises from anovel pathway of carcinogenesis dependent on dysregulation of NF-κB pathway interme-diates such as TRAF3 or CYLD.

Survival analysis based on TRAF3/CYLD status was expanded to the entire TCGAHNSCC cohort. CYLD knockdown was achieved in vitro using CRISPR/Cas9. WesternBlotting and a luciferase reporter assay were used to confirm CYLD depletion and NF-κBactivation, respectively. Parental or CYLD-depleted cells were then transfected with HPVDNA and HPV replication was determined using qRT-PCR. Finally, long control region(LCR) transcriptional activity was assessed in parental or CYLD-depleted cells using a lu-ciferase reporter assay as a correlate for HPV replication and gene expression.

We found that mutations in TRAF3 and CYLD accounted for 28% of HPV-positiveHNSCC. Patients with HPV-positive tumors harboring TRAF3/CYLD mutationsdemonstrated markedly improved survival over patients with HPV-positive tumors with-out mutations or with HPV-negative tumors. CYLD knockdown in cultured cells resultedin constitutive activation of NF-κB in vitro. Preliminary data suggested that activation ofNF-κB increased HPV replication and activity at the LCR.

Together, our data define a previously unrecognized subset of HPV-positive HNSCCthat may rely on constitutively active NF-κB. Furthermore, mutations in TRAF3 andCYLD may serve as biomarkers in therapeutic de-escalation trials for HPV-positive HN-SCC. Finally, we began establishing a cellular model that displays activation of NF-κBthrough CYLD depletion. This model will be useful to further investigate mechanismsof HPV-driven carcinogenesis in the head and neck.

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This is an Open Access Thesis.

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