Date of Award

2021

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Jianxin You

Abstract

Merkel cell polyomavirus (MCPyV) infection causes near-ubiquitous, asymptomatic infection in the skin, but occasionally leads to an aggressive skin cancer called Merkel cell carcinoma (MCC). Epidemiological evidence suggests that poorly controlled MCPyV infection may be a precursor to MCPyV-associated MCC. Improved understanding of host responses that normally control MCPyV infection could inform prophylactic measures in at-risk groups. Similarly, elucidating the sensitivities imbued by the presence of MCPyV in most MCCs could yield targeted solutions for metastatic MCC cases in which therapeutic options are limited. We performed MCPyV infections in primary human dermal fibroblasts and examined the interface between virus and host cell via several methods including qPCR, western blotting, immunofluorescence microscopy, qPCR, and lentiviral CRISPR knockout. We found that MCPyV infection leads to innate inflammatory cytokine induction in response to late events in the viral infectious cycle. This innate gene induction was mediated by the cGAS-STING and NF-κB pathways and has the potential to restrict MCPyV replication. We also screened natural compounds for their activity against the MCPyV promoter and for specific cytotoxicity in MCPyV-positive MCC cell lines. We determined the mechanism of action of a lead compound using cell viability assays, a reverse-phase protein array, and western blotting. This compound induces DNA damage and cell death specifically in MCPyV-positive MCCs. Combined treatment with BCL-2 inhibition led to more complete killing and expanded the phenotype to a broad set of MCPyV-positive MCC cell lines. Our findings provide foundational context to the events prior to MCPyV-driven oncogenesis and to the susceptibility of MCPyV-associated tumors to medical intervention. The MCPyV infection experiments illustrate the manner in which healthy cellular conditions might allow low-level infection until highly active replication is restricted by host responses. Conversely, pathologic conditions could result in unbridled MCPyV replication that licenses MCC tumorigenesis. Moreover, the profound vulnerability to DNA damage and BCL-2 inhibition shared by MCPyV-positive MCC cell lines underscores the clinical relevance of MCPyV presence in the future solutions to metastatic MCC.

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Dissertation_NKrump_EmbeddedFileLinks_2021.docx (3150 kB)

Included in

Microbiology Commons

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