SUMMARY Kaposi’s Sarcoma-associated herpesvirus (KSHV) causes the AIDS-defining cancer Kaposi’s Sarcoma (KS). The KSHV-infected KS tumor cells (KSCs) express proliferation markers, indicating a loss of contact inhibition of proliferation (CIP). CIP is considered a tumor suppressive pathway, and loss of CIP is a crucial feature of oncogenic transformation in solid tumors. How KSHV antagonizes CIP is not known. The KS tumor cells most likely originate from microvascular lymphatic endothelial cells (LECs). While LECs therefore represent a relevant model for studies of KS, KSHV-induced proliferation after de novo infection of primary human LECs has not been demonstrated. We have developed a protocol for KSHV infection of primary human LECs that allows us to measure KSHV-induced loss of CIP. The central hypothesis underlying this application is that KSHV-induced loss of CIP is a critical driving feature of oncogenesis in KS. Our preliminary work shows that the KSHV miR-K10 miRNAs contribute substantially to the KSHV-induced loss of CIP in LECs but are not the only viral determinants of this phenotype. Our results furthermore implicate viral repression of p27, disruption of adherens junctions (AJs), and deregulation of the cytoskeleton and vesicular trafficking in KSHV-mediated loss of CIP. To test our hypothesis and elucidate the mechanisms underlying KSHV-induced loss of CIP, we propose three Specific Aims. In Specific Aim 1, we will determine the expression of the four miR-K10 miRNAs in KSHV-infected LECs and KS. We will also define their individual contributions to the KSHV-induced loss of CIP. In Specific Aim 2, we will identify the mechanisms underlying the miR-K10-induced loss of CIP in KSHV- infected LECs. In Specific Aim 3, we will identify other viral genes that promote the KSHV-induced loss of CIP. The proposed study is innovative because our model provides rigorously defined experimental settings that enable the analysis of KSHV-induced loss of CIP after infection of a primary human cell type with relevance to KS. This work is significant because it will establish the viral determinants of KSHV-induced LEC proliferation, thereby explaining oncogenic mechanisms in KS. Results will be impactful since CIP is a tumor-suppressive mechanism. Understanding how KSHV overcomes CIP will help us to explain how KSHV causes KS and could potentially be exploited for therapeutic intervention in KS.

PROJECT NARRATIVE Kaposi’s Sarcoma-associated herpesvirus (KSHV) is a major cause of AIDS-associated cancers and thus represents a significant public health burden. The proposed work seeks to establish how KSHV triggers cellular proliferation in an improved cell culture model of the KSHV-associated cancer Kaposi’s sarcoma. Results will be impactful, because they will improve our understanding of mechanisms underlying Kaposi’s Sarcoma and our ability to develop therapeutic strategies for this cancer.