Expression Profiles in ATL Patients

Masison, Cynthia

Abstract

Human T-lymphotropic virus type 1 (HTLV-1) is a complex delta-retrovirus that infects fifteen to twenty million individuals worldwide. HTLV-1 is the etiological agent of adult T-cell leukemia (ATL), an aggressive and fatal disease. Although several therapeutic approaches have been used, ATL continues to carry a very poor prognosis. With the advent of DNA microarray technology, expression profiles of HTLV-1-infected cells and Tax-expressing cells have allowed a more complete list of HTLV-1/Tax regulated genes. In collaboration with Tom Waldmann's group, we examined 32 ATL patients with smoldering, chronic, lymphoma and acute leukemia using the Affymetrix HG-U133A2.0 array containing probe sets for 14,500 genes. Expression patterns revealed distinct clustering of leukemia, lymphoma and control samples. Using BRB array and PathwayStudio programs, we identified genes differentially expressed in leukemia and lymphoma cells compared to normal donor lymphocytes. Examination of PBMCs from lymphoma patients indicate changes in CSPG2 and immune response pathways characterized by high levels of genes including TLR2, CCR1, CD14, CD33, ICAM1 and MHC Class II molecules. Leukemia cells were characterized by a striking increase in genes linked to CDC2/cyclin B1, RANKL, SYK/LYN tyrosine kinases and BIRC5. Because of its potential therapeutic importance, we focused our studies on the regulation and function of BIRC5, whose expression was increased in 13 of 14 leukemia samples. TCF4 reporter assays and transfection of DN-TCF4 demonstrated that TCF4 regulates BIRC5 gene expression. Functionally, transfection of ATL cells with BIRC5 shRNA decreased BIRC5 expression and cell viability 80%. Clinical treatment of ATL patients with Zenapax or bortezomib decreased BIRC5 expression and cell viability. These experiments represent the first direct experimental evidence that BIRC5 plays an important role in ATL cell viability. This study represents a comprehensive microarray analysis of ATL patient samples and provides insight into ATL genesis and potential biomarkers and targeted therapies. To determine the direct role of the viral oncoprotein, Tax in these pathways, we have recently developed a series of reagents which allow a high percentage of transduction in different cell types and a level of Tax expression that is comparable to that of HTLV-1 cells. The lentivirus vectors allow expression of Tax protein that is tagged either at its N-Terminus with a Flag tag or at its C-Terminus with a TAP tag (Flag-Tax and Tax-TAP, respectively). The TAP(Tandem Affinity Purification) tag allows one to purify proteins and to determine their specific partners in protein-protein interaction experiments. Initial studies indicate that we are able to achieve high transduction efficiency and Tax expression in different cell types, (55 % in MOLT4 T cell line and 95% in 293T/Hela cells). Luciferase assays demonstrated that these proteins are transcriptionally active on the HTLV-1 promoter (LTR) with up to 275 fold activation, but also on an NF-kB promoter (up to 4458 fold activation). Moreover the Tax intracellular localization is similar to that previously published, i.e. with a strong nuclear staining. Gene expression profiles were then determined using GeneChip Affymetrix technology using either 293T or MOLT4 cells infected with Tax lentiviral vectors. We focused on genes that were up-regulated by 3 fold or more in Tax expressing cells, over control samples (cells infected with empty vector). As a validation of our system, we found several genes that have already been described as being upregulated in HTLV-1 infected cells including: the anti-apoptotic BIRC-3/CIAP-2, the tumor suppressor gene EGR-1, the cell surface receptor protein of the TNF receptor family FAS, the growth arrest and DNA-damage-inducible beta GADD45B, the Inter-Cellular Adhesion Molecule 1 ICAM-1/CD54, interleukin 15, JAK kinase 3 and the proto-oncogenes JUN-B and REL. More interestingly, we found novel gene targets. One gene upregulated in Tax expressing cells was Gem. Gem is a member of the small GTP-binding proteins within the Ras superfamily, referred as the RGK (for Rad, Gem and the mouse orthologue, Kir. Gem, a gene that was first identified in mitogen-induced human peripheral blood T cells was up-regulated up to 28 fold in Tax expressing cells. In addition to an increase in mRNA levels, Gem protein was also overexpressed in Tax transduced cells, while it was absent in mock infected cells. Importantly, Gem protein was expressed at high levels in HTLV-1 infected T cell lines (C8166, C91/PL, HUT102 and MT2) or HTLV-1 infected dendritic cells but not uninfected control cells. Further, using luciferase reporter and chromatin immunoprecipitation assays, we found that Tax activates transcription of the Gem promoter through CRE elements. Ongoing studies are investigation the role of Gem in HTLV-1 pathogenesis. Using the information we gained from the microarray studies on ATL, we have established several collaborations investigating gene expression patterns in a variety of human cancers. Our collaboration with Dr. Michael Birrer has yielded important results on ovarian cancer. Despite the existence of morphologically indistinguishable disease, patients with advanced ovarian tumors display a broad range of survival end points. To resolve survival-associated loci, gene expression profiling was completed for an extensive set of 185 (90 optimal/95 suboptimal) primary ovarian tumors using the Affymetrix human U133A microarray. The prognostic signature successfully classified the tumors according to survival for suboptimally but not optimally debulked patients. The suboptimal gene signature was validated using the independent set of tumors. To elucidate signaling events amenable to therapeutic intervention in suboptimally debulked patients, pathway analysis was completed for the top 57 survival-associated probe sets. For suboptimally debulked patients, confirmation of the predictive gene signature supports the existence of a clinically relevant predictor, as well as the possibility of novel therapeutic opportunities. In conjunction with Dr. Giovanna Tosatos laboratory, we examined the contribution of ORFK13/vFLIP to the Kaposi's sarcoma (KS) phenotype and potential for therapeutic targeting. Retroviral transduction of ORFK13/vFLIP into primary human endothelial cells induces the spindle morphology distinctive of KS cells and promoted the formation of abnormal vascular networks;upregulated the expression of proinflammatory cytokines, chemokines, and interferon-responsive genes;and stimulated the adhesion of inflammatory cells characteristic of KS lesions. Expression profiles revealed that the cellular enzyme thymidine phosphorylase (Pd-Ecgf) was markedly induced by ORFK13/vFLIP. Pd-Ecgf can metabolize the prodrug 5-fluoro-5-deoxyuridine (5-dFUrd) to 5-fluouridine (5-FU), a potent thymidine synthase inhibitor, which blocks DNA and RNA synthesis. When tested for cytotoxicity, 5-dFUrd (0.1 to 1 microM) selectively killed ORFK13/vFLIP-expressing endothelial cells while sparing control cells. These results demonstrate that ORFK13/vFLIP directly and indirectly contributes to the inflammatory and vascular phenotype of KS and identify 5-dFUrd as a potential new drug that targets KSHV latency for the treatment of KS and other KSHV-associated malignancies.