A Role for KSHV in the Pathogenesis of Malignancies

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We have focused primarily on the study of primary effusion lymphoma in patients with AIDS. Primary effusion lymphoma (PEL) is a Kaposi's sarcoma herpes virus (KSHV)-induced lymphoma that typically arises in body cavities of HIV-infected patients. PEL cells are often co-infected with Epstein-Barr virus (EBV). "PEL-like" lymphoma is a KSHV-unrelated lymphoma that arises in body cavities of HIV-negative patients. "PEL-like lymphoma" is sometimes EBV-positive. The derivation of PEL/"PEL-like" cells is unclear. To study PEL pathogenesis, mesothelial cells were cultured from body cavity effusions of 23 patients. These included patients with AIDS with or without PEL presenting with effusions in body cavities, and patients with ovarian carcinoma with malignant or not malignant effusions. Cell proliferation, cytokine secretion, marker phenotypes, KSHV/EBV infection and clonality were evaluated. Gene expression was measured by qPCR and immunoblotting. A mouse model of PEL was used to evaluate tumorigenicity. We found that mesothelia derived from 6 effusions of HIV-infected patients with PEL or other KSHV-associated diseases contain rare KSHV+ or EBV+ mesothelial cells. After extended culture (16-17 weeks), some mesothelial cells underwent a trans-differentiation process generating lymphoid-type CD45+/B220+, CD5+, CD27+, CD43+, CD11c+ and CD3- cells resembling "B1-cells", most commonly found in mouse body cavities. These "B1-like" cells were short-lived. However, long-term KSHV+EBV- and EBV+KSHV- clonal cell lines emerged from mesothelial cultures from two patients, which were clonally distinct from the monoclonal or polyclonal B-cell populations found in the patient's original effusion. The current study provides a novel and unifying insight into PEL and "PEL-like" lymphomagenesis. Three observations emerged from these studies. First, we discovered that mesothelial cell monolayers undergo a "mesothelial-to-lymphoid" transition (MLT)" resulting in the emergence of "lymphoid-type" cells. This discovery extends the spectrum of mesothelial functional capabilities, beyond secretion of lubricants, maintenance of surface integrity and ability to repair. MLT resembles the emergence of hematopoietic cells from the endothelium of the yolk sac and the dorsal aorta identified as "endothelial-to-hematopoietic transition" (EHT). Endothelial and mesothelial cells can undergo phenotypic and functional change through "endothelial-to-mesenchymal" (EMT) and "mesothelial-to-mesenchymal" transitions (MMT). The second discovery we made is that the lymphocytes emerging from mesothelial cultures have a "B1-like" phenotype, supporting a mesothelial origin of human "B1-type" cells. B1 lymphocytes are the main B-cell population in murine body cavities but are rare elsewhere. Human "B1-like" lymphocytes have been identified in cord and peripheral blood, but to our knowledge not in body cavities. Despite their importance in immune defense, the origin of "B1-type" cells is unclear. During mouse development, B1 lymphocytes are first detected at embryonic day (E) 8.0-8.5 in the para-aortic mesoderm prior to the emergence of hematopoietic stem cells (HSC) from the dorsal aorta, suggesting an HSC-independent origin of B1 cells. All mesothelia that line body cavities derive from the para-aortic splanchnopleural mesoderm. Thus, "B1-type" cells and mesothelia have a common developmental derivation, raising the possibility that persistence of mesodermal precursors within adult mesothelia confers B1-cell differentiation potential to these mesothelia. The third observation we made is that mesothelial cells can be infected by KSHV and EBV. The emergence of monoclonal B-lineage cell lines from mesothelial cultures suggests that PEL and PEL-like lymphoma may derive from KSHV or EBV-infected mesothelial cells. It is noteworthy that two of the 3 lines so derived were clonally distinguishable from the cells originally found in the patient. It remains possible that these lines may have emerged as a result of outgrowth from rare clones of PEL or EBV-infected cells in the original effusion. However, the evidence of MLT transition and clonal analysis suggest that a more likely explanation is that the unique clonal lines derived from the KSHV or EBV-infected mesothelial cells. Interestingly, the "indeterminate" surface phenotype of PEL cells and cell lines, including the KSHV+ 81 lines, resembles the predominant surface phenotype of the "B1-like" cells. In addition, the monoclonal KSHV-/EBV+ clone resembles the KSHV- "PEL-like" lymphoma in showing a mature B-cell phenotype and EBV infection, raising the possibility that the CD20+ cell subset recovered from mesothelial cultures is a source of "PEL-like lymphoma". From this perspective, body cavity-associated PEL and "PEL-like" malignancies would have a common mesothelial derivation. Ongoing and future studies will be focused on determining if this new understanding of PEL pathogenesis can be exploited to prevent PEL development and treat it by targeting the mesothelium. To pursue this goal, we are developing a mouse model in which cultures of human mesothelium infected or not infected with KSHV are explanted into immunodeficient mice. We want to observe the localization of peritoneal explants of mesothelium, its natural history in the mouse body cavity, the potential emergence of "B1"-type cells and the potential emergence of PEL. Since inflammation within the body cavities is commonly detected in patients with AIDS, we will explore the role of experimental inflammation in the emergence of human B1-type cells and PEL. We have obtained engraftment of PEL-derived primary mesothelial cells expanded in culture and transferred into the peritoneal cavity of immunodeficient mice (NSG mice). Macroscopic examination of the peritoneal cavity showed the presence of focal nodular structures and more diffuse thickening of the peritoneum. We did not detect the presence of PEL-type cells floating in the peritoneal cavity. We are currently further evaluating these lesions and their relationship with KSHV diseases. Following up on our previous laboratory studies highlighting the role of VEGFA in the pathogenesis of KS, we have collaborated with the clinical group of HAMB in a pilot study of the anti-VEGF antibody Bevazizumab combined with liposomal doxorubicin in a group of patients with advanced KS. Evaluation of this clinical study showed that patients with advanced KS are responsive to the combination of liposomal doxorubicin and Bevazizumab. Future studies will need to directly assess if the drug combination yields a better outcome than liposomal doxorubicin alone in patients with advanced KS. In a collaborative study, we have examined further the role of viral IL-6 in HSHV infection. We have now discovered that vIL-6 epigenetically silences caveolin-1 (CAV1) expression to promote angiogenesis and tumorigenesis by regulating the formation of STAT3-DNMT1 complex. This new information will guide future studies on epigenetic changed induced by KHSV infection of endothelial cells. Recently, we have examined at a fundamental level the role of DLC1 deficiency in KHSV-associated malignancies with a focus on KS. Here, we have generated mouse lines with inducible and endothelial-specific DLC1 deficiency, and preliminarily identified DLC1 deficiency as a potential driver of endothelial growth following KHSV infection.