Evidence of an Oncogenic Role for Aberrant Tox Activation in Cutaneous T Cell Lymphoma

Cutaneous T cell lymphoma (CTCL) consists of two main subtypes: mycosis fungoides (MF), which primarily affects the skin; and Sézary syndrome (SS), which is characterized by the presence of circulating malignant Sézary cells. Although defective apoptosis and cell cycle progression have been implicated in CTCL disease development, the precise molecular mechanism underlying these abnormalities is only partially understood. Recently our group showed that TOX, a transcription factor transiently expressed during thymocyte development, was ectopically expressed in the malignant CD4⁺ T cells in MF skin lesions. Normally in mature CD4⁺ T cells, TOX is expressed at very low levels. Not only is TOX highly expressed in the infiltrating CD4⁺ T cells in MF skin lesions, but its expression levels also inversely correlate with the clinical outcome of CTCL patients. However, its involvement in other types of CTCL remains unknown and its biological effects on CTCL pathogenesis have not been explored. In this study, we sought to examine if aberrant TOX expression occurs in SS (a leukemic variant of CTCL), and whether it contributes to CTCL pathogenesis. We found that TOX expression is also enhanced significantly in primary CD4⁺CD7^- cells from SS patients (n=12), and its enhanced transcript levels correlate with higher disease-specific mortality (P=0.039). To investigate the role of TOX in CTCL, we corrected the ectopic TOX expression by lentiviral-shRNA-mediated TOX gene knock-down in CTCL cell lines (Hut78, HH, and SZ4). In all three cell lines, this resulted in markedly increased apoptosis (measured using Annexin V apoptosis assay, 2-3 fold, P<0.05) and reduced cellular proliferation (assessed by BrdU incorporation assay and colony formation in 3D cultures, 1.5-12 fold, P<0.05). In line with these phenotypes, TOX knock-down led to an increase in cleaved caspases (3, 9) and enhanced expression of multiple cell cycle inhibitors (CDKN1B, CDKN1C). Moreover, to test the effect of TOX suppression on the tumor-inducing ability of CTCL cells in vivo, we injected TOX-suppressed cells or control CTCL cells (1x10⁶ per injection site) subcutaneously into NOD-SCID IL2rγ null (NSG) mice and monitored local tumor formation. Control CTCL cells (Hut78, HH) induced local tumor growth in six out of six mice within 2 weeks of injection, with enlargement progressively to 1.5cm in diameter within 4 weeks of injection. In contrast, TOX silencing effectively reduced the tumor volume induced by HH cells (n=6) and abolished the tumor-forming ability of Hut78 cells (n=6). Since TOX is a DNA-binding nuclear protein, we next sought to explore the potential downstream molecular partners of TOX by performing comparative transcriptome analysis between TOX-suppressed cells and control CTCL cells (Hut78, HH). The expression of several tumor suppressor genes, including SMAD3 and FOXO3, was increased upon TOX knock-down in both Hut78 and HH cell lines. Pathway analyses of the differentially-expressed genes uncovered activation of apoptotic pathways and cell cycle arrest processes upon TOX suppression. These findings were validated using quantitative RT-PCR. In conclusion, our results indicate that ectopic expression of TOX in CTCL cells causes the proliferation/apoptosis balance to shift toward proliferation through suppression of the transcription of multiple tumor suppressors. Therefore abolishing TOX activity may be a promising treatment strategy for CTCL.