HPV Attenuation of the cGAS/STING Pathway

Project Summary Globally, human papilloma virus (HPV) causes 5% of cancers and 30% of head and neck squamous cell carcinomas (HNSCC). While HPV is the most common sexually transmitted infection, most infections are cleared by the innate immune system. Only long-term infections, occurring when the virus evades the innate immune

system, can become cancerous. HPV oncoproteins, specifically E6 and E7, may increase HPV persistence by disrupting innate immune pathways such as cGAS/STING. The cGAS/STING pathway senses cytosolic double stranded DNA (dsDNA) and initiates a type I interferon (IFN) response, which can promote immune clearance

of the virus. Upon sensing dsDNA, cGAS combines ATP and GTP to generate cGAMP which activates STING, ultimately resulting in the production of type I IFNs. Given that IFN has antiviral and antitumor properties, HPV likely evolved to counteract this pathway. Indeed, there is evidence that HPV oncogenes evade and manipulate

the cGAS/STING pathway. However, HPV may generate several different types of cytosolic DNA; it can induce genomic DNA damage, may be shuttling viral DNA into the cytosol, and has been correlated to increased mitochondrial DNA copy number. While these damage associated molecular patterns can activate the

cGAS/STING/IFN pathway, this has not been demonstrated in the case of HPV infection. Despite uncertainty as to the source of cGAS/STING pathway activation in HPV(+) cells, pathway suppression is clearly important. Our preliminary data, working with full-length and recombinant HPV genomes in primary patient-derived keratinocytes, indicates that HPV infection reduces the activation of the

cGAS/STING/IFN pathway when exposed to exogenous DNA. I demonstrate that HPV E6 expression promotes STING degradation. In parallel, I demonstrate that, despite interfering with pathway activation, HPV(+) tonsil cells have increased baseline cGAMP production compared to HPV(-) patient-matched tonsil cells, raising the

question of what is activating cGAS in HPV(+) cells. I hypothesize that HPV causes genomic DNA damage leading to the production of cGAMP, and to circumvent this, E6 promotes STING degradation. To test this hypothesis, I will determine the mechanisms by which high-risk HPV E6 drives STING degradation as well as identifying the source of nucleic acids causing cGAS activation in HPV(+) cells. Aim 1 of

this proposal will assess the rate of STING degradation, how it is impacted by HPV E6, and the contribution of two E3 ubiquitin ligases on STING degradation. Aim 2 will identify the source of cGAS-activating nucleic acids present in HPV(+) tonsil cells and HPV(+) head and neck cancer tumors as well as the contribution of

mitochondrial and genomic DNA damage on cGAS activation. Completion of the proposed studies will provide greater understanding of how the HPV oncogene E6 impacts STING degradation. It will also identify the source of nucleic acids activating cGAS in HPV(+) tonsil cells. Additionally, this work will provide me with valuable tools

and training to prepare me for a career in immunology, virology, and cancer research after graduate school.