Gene expression profile for KSHV infection and methods for treating same

#Kaposi's Sarcoma (KS) is a vascular neoplasm characterized by angioproliferative lesions on the skin and visceral organs. The lesions are distinguished by the presence of proliferating spindle-shaped tumor cells of endothelial origin and exhibit abnormal vascularization with extensive extravasation of inflammatory cells and erythrocytes. While KS is a rare condition in immunocompetent individuals, it is the most common malignancy associated with acquired immunodeficiency syndrome (AIDS). Human herpesvirus 8 (HHV8) or Kaposi's sarcoma-associated herpesvirus (KSHV) is the infectious agent associated with KS development(14). The virus infects the majority of spindle cells in the lesion, as well as lesional endothelial cells and infiltrating leukocytes(78, 79, 80, 81). The majority of spindle cells harbor the KSHV genome in latent form with a small percentage of cells entering lytic cycle and producing infections virus(72, 82, 80, 81). The exact role of latent and lytic virus gene expression in the complex biology of KSHV has yet to be fully elucidated. In addition, little is known about the influence of virus gene expression on cellular gene profiles or how these virus-cell interactions contribute to tumorigenesis. The establishment of valid in vitro culture systems should allow resolution of many questions associated with KSHV and KS. We recently developed such a culture system using immortalized dermal microvascular endothelial cells that are extremely permissive for KSHV infection and maintain the genome indefinitely in a primarily latent state. Infection of DMVEC with KSHV leads to phenotypic changes including spindle cell formation, loss of contact inhibition and growth in soft agar, suggesting virus-cell interactions that mimic those driving KS lesion formation in vivo.

#The changes induced by KSHV infection are reflected in an altered transcriptional program of the host cell. DNA microarray analysis enables the contemporaneous monitoring of transcriptional changes for several thousands of host genes(60). Automated quantitation of RNA samples hybridized to glass slides imprinted with known DNA fragments allows for comparison of transcriptional differences under various experimental conditions.

#Kaposi's Sarcoma-associated herpesvirus infection of human endothelial cells leads to spindle cell formation and malignant transformation. DNA microarray analysis demonstrated that virus infection caused profound changes of multiple cellular pathways including those governing angiogenesis, inflammation and differentiation. Upon induction of the lytic cycle, KSHV further modulated the host cell response. To evaluate whether genes overexpressed during the lytic cycle constitute potential antiviral targets, we systematically inhibited their induction or function. Haloperidol, an antagonist of the type I Sigma receptor, and Ht-31, a peptide inhibitor of the interaction between kinase anchoring proteins (AKAP) and protein kinase A (PKA), inhibited expression of lytic, but not latent viral gene expression. One of the genes significantly up regulated following KSHV infection was the gene for the c-Kit (CD 117) protein, a receptor tyrosine kinase that functions as the receptor for stem cell factor (steel factor, SCF)(83, 84). KSHV-infected DMVEC exhibit an enhanced proliferative responsive to exogenous SCF and inhibition of c-Kit signalling leads to a reduction in ligand-dependent proliferation and reverses the KSHV-induced transformed phenotype. Furthermore, inhibition of c-kit signal transduction prevents KSHV-induced spindle cell formation whereas adenovirus vector-mediated expression of c-kit induces spindle cell formation in uninfected endothelial cells. Thus, KSHV contributes to KS development through modulation of c-Kit expression and function. The present invention provides methods and compositions for treating and diagnosing KSHV infection, as well as Kaposi's Sarcoma. The present invention also provides the gene expression profile for KSHV infected cells at various stages of viral replication. In addition, the present invention provides methods and compositions for the treatment and diagnosis of endothelial cell transformation. The present invention also provides methods and compositions for modulating angiogenesis, including to stimulate or inhibit neo-vascularization, as well as modulating the extracellular matrix surrounding endothelial cells. The present invention provides methods and compositions for the modulation of interferon mediated gene expression. Additionally, the present invention provides methods and compositions for the modulation of endothelial cell inflammation. Also included in the present invention are methods and compositions for the dedifferentiation of endothelial cells. The present invention is also directed to methods of using said gene expression profiles and microarrays, and business methods directed to said use of gene expression profiles and microarrays.

#FIG. 1, Panels A, B and C: Induction of c-Kit mRNA and protein expression on DMVEC following KSHV infection.

#A) RT-PCR for c-Kit mRNA using reverse transcribed total RNA prepared from mock-(Mock) and KSHV-infected (KSHV) DMVEC.

#RT-PCR reactions were performed using primers designed to amplify a 242 bp fragment of c-Kit. RT-PCR for HPRT was performed as a control for each sample. Amplification products were visualized by ethidium bromide staining of agarose gels,

#The analysis of host cell-virus interactions by DNA microarrays allows for detection of previously unknown relationships between cellular pathways and viral infection. By employing this technology to endothelial cells harboring KSHV, we identified cellular genes whose expression is regulated by KSHV. One of the cellular transcripts that was consistently upregulated in all three experiments was c-kit. C-kit is a receptor tyrosine kinase activated by stem cell factor (SCF). c-Kit over expression following KSHV infection affects both paracrine and autocrine c-Kit/SCF interactions. The enhanced expression with functional consequences raises the possibility that c-Kit expression may play a role in the development of KS. KSHV-infection of DMVEC enhances expression of the SCF receptor, c-Kit, and increased expression promotes enhanced proliferation in response to exogenous SCF. Further, DMVEC co-express the c-Kit ligand SCF, and that inhibition of autocrine c-Kit tyrosine kinase activity inhibits the transformed phenotype induced in DMVEC by KSHV infection. While c-Kit expression and c-Kit/SCF interactions are crucial for the normal development of hematopoietic cells and a restricted number of non-hematopoietic cells, expression of c-Kit has also been associated with various malignancies. In c-Kit-associated cancers, different roles for c-Kit have been identified. For example, in GISTs and germ cell neoplasms, gain-of function mutations in juxtamembrane and tyrosine kinase domains have been described, permitting, respectively, ligand-independent dimerization or constitutive activation without dimerization(85, 86, 87, 88). In small cell lung carcinoma (SCLC) and breast cancer, co-expression of c-Kit and SCF occurs and is thought to generate an autocrine growth loop(89, 90, 91, 92, 93). Since endothelial cells are the precursors of KS spindle cells, our finding that KSHV infection of dermal endothelial cells enhances c-Kit expression with functional consequences raises the possibility that c-Kit expression may play a role in the development of KS. A recent report documented for the first time expression of c-Kit in KS tissue(94) and, in one case examined in more detail, no activating mutations in juxtamembrane or tyrosine kinase domains were observed. This in vivo finding supports that c-Kit plays a role in the pathogenesis of KS.

#During KS disease, the presence of KSHV in a typical endothelial cells in KS lesions suggests a role for KSHV in spindle cell development and tumor progression. In our study, the observation that KSHV infection of DMVEC up-regulates functional c-Kit receptors, combined with the demonstration that c-Kit activity is required for cellular transformation, strongly supports a role for c-Kit/SCF in the development of KS.

#Identification of a causative role for c-Kit in KSHV-associated cellular transformation suggests a novel therapeutic target for KS. STI 571, the c-Kit tyrosine kinase inhibitor described in this report, has shown considerable promise in phase I/II clinical trials for the treatment of chronic myelogenous leukemia(95), a disease associated with Bcr-Abl kinase activity. Recently, STI 571 was also shown to inhibit the growth of SCLC cell lines in vitro via inhibition of c-Kit activity(93, 96). Over 70% of SCLC co-express c-Kit and SCF, suggesting that inhibitors of c-Kit activity may have clinical application for treatment of this and other cancers in which autocrine c-Kit signaling plays a role. In conclusion, c-Kit should be considered a primary target in KS tumorigenesis. Consequently, STI 571, or other pharmacological inhibitors of c-Kit signaling, should be evaluated as potential therapeutic agents for the treatment of KS.