Summary Merkel cell polyomavirus (MCPyV) is a clear cause of Merkel cell carcinoma (MCC), a highly lethal skin cancer. MCPyV encodes a small T antigen (ST) that is essential for MCC, capable of transforming cells and interacting with protein phosphatase 2A (PP2A). This Project will search for novel insights into the mechanisms by which MCPyV contributes to MCC. A critical barrier to our understanding of how MCPyV contributes to the development of MCC is the lack of appreciation of whether MCPyV T antigens differ in their transforming activities from the canonical SV40 and murine polyomavirus T antigens. A second critical barrier is whether MCPyV ST itself and its interaction with PP2A are required in the development of MCC. A third critical barrier to our understanding of how MCPyV T antigens contribute to oncogenesis is the uncertainty regarding the cell- of-origin for MCC. To address these goals we propose the following Aims. Aim 1. Assess how MCPyV ST activates MTOR signaling pathways to promote transformation. The MTOR pathway is frequently activated in MCPyV-positive MCC. Furthermore, MTOR activation appears to contribute to survival of MCC tumors. Several reports have indicated that MCPyV ST can activate the MTOR downstream targets 4EBP1 and S6K although how it does this is not well understood. We have evidence that MCPyV ST induces expression of certain amino acid transporters. We propose that this effect contributes to activation of MTOR signaling especially during amino acid starvation. In addition, we have observed that MCPyV ST can specifically increase levels of lactate transporters reflecting an increase of intracellular lactate levels and may activate NFκB signaling. We will determine whether increased levels of the amino acid and lactate transporters contribute to MCPyV ST- mediated transformation. Aim 2. Determine the mechanism of inhibition of double stranded DNA break repair by MCPyV ST. We have observed that MCPyV ST can inhibit double stranded DNA break (DSB) repair in response to ionizing radiation (IR). We have also observed that MCPyV ST specifically inhibits non-homologous end joining (NHEJ) by interfering with the activation of DNAPK. We will determine if the ability of MCPyV ST to inhibit DSB repair by perturbing DNAPK and NHEJ activity is linked to perturbation of PP2A. We will determine the specific role of PP2A that is required by MCPyV ST to inhibit DNAPK and NHEJ activation and if this newly described property of MCPyV ST contributes to its overall transforming functions. Aim 3. Test the behavior of MCPyV T Antigens in Merkel cells. We have pursued isolation of primary human Merkel and precursor cells to determine if they are sensitive to transformation by MCPyV T antigens. We will establish an in vitro transformation system using Merkel cells or their precursors to test the role of the MCPyV T antigens in their natural background and clarify the specific oncogenic driver events required to generate an MCC tumor...