Molecular mechanisms governing the ubiquitination signaling during KSHV cell entry and tumorigenesis

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Infection with Kaposi sarcoma herpesvirus (KSHV) has been shown to increase the risk of classic Kaposi's sarcoma (KS), AIDS-associated KS (AIDS-KS) and lymphoproliferative disorders, including primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD) in HIV/AIDS. About 30-40% of AIDS-related cancers in HIV+ individuals treated with antiretroviral drugs are caused due to KSHV infection, and in many circumstances they are incurable. Emerging studies have indicated that chronic inflammation mediated by transcription factors, including STAT3 and NF-κB, in KSHV-infected cells plays critical role in the development of KSHV-associated malignancies in HIV (+) individuals. Protein ubiquitination, a post-translational modification, plays key roles in the regulation of NF-κB and STAT3 activation, pathogenic virus entry into target cells, and survival of virus infected cells. Using an unbiased Ubi-Scan proteomics approach, we have identified tumor specific CADM1 ubiquitination at lysine 437 (K437), which is essential for chronic activation of STAT3 and NF- κB and the proliferation and survival of PEL cells. CADM1-K63-Ubi is also critical for efficient KSHV entry into target cells after KSHV de novo infection. Using siRNA library-based screening we identified the E2 ubiquitin conjugating enzyme Ubc13 and the E3 ubiquitin ligase Mib1, as essential factors for CADM1 ubiquitination. In addition, we found that ubiquitinated CADM1 was required for the activation of the small GTPase Rac1 in KSHV- infected cells, which in turn was critical for chronic STAT3 and NF-κB activation and PEL cell proliferation and survival. Furthermore, using siRNA library-based screening of Guanine nucleotide exchange factors (GEFs), we have also determined that the GEF TIAM1 is required for Rac1 activation. Therefore, we hypothesize that KSHV de novo infection-induced rapid Ubc13 and Mib1-dependent CADM1-K63 ubiquitination leads to the activation of TIAM1/Rac1 and NF-κB and STAT3 essential for KSHV entry into target cells, KSHV latency establishment and KSHV lymphoma cells survival and proliferation. To test this hypothesis, we propose three specific Aims: 1) Mechanistic roles of CADM1-K63-Ubi and its associated molecules in KSHV entry into target cells after de novo infection, 2) Mechanistic roles of CADM1-K63-Ubi and its associated molecules in NF-κB and STAT3 activation after KSHV de novo infection, and 3) Examine the efficacy of small molecule inhibitors against CADM1-K63-Ubi and its associated molecules using PEL patient-derived xenograft (PDX) mouse model. Data obtained from this project will reveal the mechanisms of chronic STAT3 and NF-κB activation and efficient KSHV entry into target cells after KSHV de novo infection. These findings will pave the way for therapeutic intervention of KSHV- mediated highly aggressive AIDS associated malignancies, which are often seen throughout the US, particularly South Florida, which has one of the highest incidences of HIV in the country.