PROJECT SUMMARY Kaposi’s Sarcoma-associated herpesvirus (KSHV) causes the AIDS-defining cancer Kaposi’s Sarcoma (KS). How KSHV infection causes KS is poorly understood, primarily due to a lack of a rigorously defined primary human cell culture model that recapitulates the transformed features of the KSHV-infected tumor cells in KS. KS tumor cells most likely originate from microvascular lymphatic endothelial cells (LECs). LECs, therefore, represent a physiologically relevant model for studies of KS. We have developed a protocol for KSHV infection of primary human LECs that allows us to measure KSHV-induced loss of contact inhibition of proliferation (CIP). Loss of CIP is a feature of oncogenic transformation in solid tumors. Based on these data, our central hypothesis is that the KSHV-mediated deregulation of cellular gene expression triggers the oncogenic transformation of LECs, which recapitulates KSHV-mediated oncogenesis in KS. This proposal, therefore, aims to identify KSHV-induced genes that promote the KSHV-induced loss of CIP in KLECs. We also aim to determine if KSHV-infected LECs (KLECs) are fully transformed and form xenograft tumors in immunodeficient mice. To test our hypothesis and achieve our objectives, we propose two Specific Aims. In Specific Aim 1, we will conduct sub-genomic CRISPR screens to identify which KSHV-induced human genes promote KSHV- induced loss of CIP in KLECs. Specific Aim 2 will determine whether KLECs are fully transformed and can form xenograft tumors in immunodeficient mice upon subcutaneous or subrenal implantation. We will assess resulting tumors for the expression of viral and cellular markers of KS to determine how closely KLEC tumors model KS. The proposed study is innovative because our model provides a set of rigorously defined experimental settings that enable oncogenic changes after KSHV infection of a primary human cell type relevant to KS. This work is significant because it will identify KSHV-induced cellular genes that promote these changes and establish whether KSHV-infected LECs are fully transformed and tumorigenic in mice. Furthermore, results will be impactful because they may enable mechanistic insights and provide proof of concept that KLECs undergo oncogenic transformation. Results would also establish a well-standardized primary human cell-based model of KS in vitro and, likely, in vivo. Such a model would enable countless studies of the roles of viral and cellular genes in KSHV-mediated tumorigenesis, the identification of KS biomarkers, and the pre-clinical testing of therapeutic approaches.