VIRAL-INDUCED TUMORIGENESIS: THE CHARACTERIZATION OF AVIAN LEUKOSIS VIRUS-INDUCED NEOPLASMS BY HIGH-THROUGHPUT SEQUENCING
Justice IV, James Franklin
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Avian leukosis virus (ALV) is a simple retrovirus that infects chicken and integrates its DNA provirus into the host genome. Chickens exposed to either ALV subgroup-A (ALV-A) or ALV subgroup-J (ALV-J) during embryogenesis often develop tumors within a few months after hatching. Those exposed to ALV-A typically develop B-cell lymphomas, while birds exposed to ALV-J develop tumors of myeloid origin and hemangiomas. ALV-induced tumors have been the subject of numerous studies over the last thirty years. These neoplasms have been shown to develop via insertional mutagenesis, where the virus acts as a mutagen by integrating into the chicken genome and perturbing the expression of genes near the integration site. Early studies have located several common proviral integration sites which harbor genes thought to drive these tumors. In this thesis, I analyze ALV-induced tumors with high-throughput sequencing, which enables a much more detailed view of the integration landscape than was possible with previous techniques. First, we infect 5- and 10-day chicken embryos with ALV-A to induce rapid-onset B-cell lymphomas, and then locate viral integration sites with the Illumina Hi-Seq. Four genes—MYC, MYB, Mir-155 and TERT—have previously been identified as common integration sites in ALV-A induced tumors. We identify these genes, and implicate additional genes as drivers of tumorigenesis in these neoplasms, including TNFRSF1A, MEF2C, CTDSPL, TAB2, RUNX1, MLL5, CXorf57, and BACH2. We also observe increased frequency of ALV integration near transcription start sites and within transcripts, as well as a weak integration site consensus sequence. In addition, we studied ALV-J induced hemangiomas. We induced tumors by infecting 5-day chicken embryos with ALV-J strain PDRC-59831, a newly studied US isolate of ALV-J, which induced myeloid leukosis and hemangiomas. We sequenced integration sites and found expanded clones with integrations in the MET gene in two of the five hemangiomas studied, a gene which has not been implicated in ALV-J induced neoplasms to date. MET is a known proto-oncogene that acts through a diverse set of signaling pathways and is involved in many types of neoplasms. We show that tumors harboring MET integrations exhibit strong overexpression of the MET mRNA.