Cancer cells depend on reprogrammed metabolic pathways for anabolic proliferation. Discovering these cancer metabolic vulnerabilities can reveal novel targets for therapy. Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causal agent of Kaposi’s sarcoma (KS) and several other cancers. Despite intensive studies for several decades, there is currently no effective therapy for KS. Our long-term goal is to delineate the pathogenesis of KSHV-induced cancers, providing a scientific basis for developing novel therapies. Toward this goal, we have previously developed an efficient system of KSHV-induced cellular transformation of primary cells and a reverse genetics system for KSHV mutagenesis. Using these powerful systems, in the current funding period, we have delineated viral and cellular genes that are essential for KSHV-induced cellular transformation and identified novel therapeutic targets and agents that have the potentials for translating into clinics. In particular, we have recently discovered that KSHV-transformed cells are addicted to glutamine. Unlike other types of cancer cells that utilize glutamine to replenish the TCA cycle, glutamine is primarily shunted to nucleotides syntheses by providing the critical g-nitrogen in addition to amino acids. KSHV hijacks numerous rate-limiting enzymes in these pathways, including phosphoribosyl pyrophosphate amidotransferase (PPAT) and phosphoribosyl pyrophosphate synthetases 1 (PRPS1), which is upregulated in KS spindle tumor cells. Significantly, knockdown of these enzymes suppresses the proliferation of KSHV-transformed cells but has no effect on the primary/uninfected cells. Our hypothesis is that KSHV encodes specific gene(s) to hijack the nucleotide synthesis pathways to support the proliferation and survival of KSHV-transformed cells, and hence targeting these pathways is effective for therapy of KSHV-induced tumors. We have developed 3D Culture systems that closely representing in vivo metabolic changes, an innovative nanoparticles carbon-dots (Cdots)- mediated delivery approach for locked nucleic acid (LNA)-siRNAs, and cutting-edge technology of metabolomics for tracing the carbon and nitrogen flows. We will determine the essential roles of the dysregulated nucleotide synthesis pathways for KSHV-induced cellular transformation and tumorigenesis (Aim 1); determine the mechanisms by which KSHV hijacks the nucleotide synthesis pathways for supporting the proliferation and survival of KSHV-transformed cells (Aim 2); and target vulnerable genes in the nucleotide pathways using the Cdots-mediated delivery approach for treating KSHV-induced tumorigenesis (Aim 3). The proposed project is highly significant as it will test a novel hypothesis of KSHV manipulation of key cellular metabolic pathways using multidisciplinary innovative approaches and model systems. It is our expectation that the accomplishment of this project will lead to the identification of novel cancer vulnerabilities of KSHV-induce...