Epigenetic Disruption of ZEB1 Binding Causes Constitutive Activation of IL-15 in Cutaneous T-Cell Lymphoma

Cutaneous T-cell lymphoma (CTCL) is a non-Hodgkin’s lymphoma of skin-homing T-cells that represents 70-80% of all cutaneous lymphomas. Studies have shown that interleukin-15 (IL-15) plays a key role in T-cell oncogenesis by increasing T-cell survival, proliferation, migration and invasion in patient-derived CTCL cell lines.  Previously, we have demonstrated that IL-15 is overexpressed in lesional biopsies and peripheral blood of CTCL patients (Mishra et al., Blood, 122:1826, 2013).  However, the underlying mechanisms of IL-15 deregulation are largely unknown.  Since epigenetic modifications such as DNA methylation can alter gene expression, we selected samples highly enriched in neoplastic T-cells from the blood of CTCL patients and performed a pyro-sequencing analysis to determine methylation of the IL-15 gene promoter. We observed significantly higher methylation in the IL-15 promoter of CD4+ T-cells from CTCL patients vs. normal donors (mean ± SEM of relative % methylation= 232.0 ± 49.18, n=9 vs 100.1 ± 7.619, n=6, P=0.03). Furthermore, methylation at the IL-15 promoter was higher in malignant CD4+ T-cells compared to non-malignant neutrophils from the same patient (mean ± SEM of relative % methylation = 232.0 ± 49.18 vs 52.31 ± 6.28%, N=9 each, paired t-test, P=0.0025).  Using this approach, we also analyzed methylation of each ‘CpG dinucleotide’ in the IL-15 promoter. Once again, methylation was higher in CD4+ T-cells of patient versus normal donors and, within each patient, in CD4+ T-cells versus neutrophils. The CpG rich region of the human IL-15 promoter contains three putative binding sites for the known transcriptional repressor Zeb1, which has been reported to be mutated or deleted in CTCL. In order to examine the effect of CpG methylation at the Zeb1 binding region on IL-15 transcription, the CpG rich 5’ regulatory region of the IL-15 promoter was amplified and cloned into PGL3 luciferase vector. As measured by relative luciferase activity, the region of the IL-15 promoter containing the 3 ZEB1-binding sites is transcriptionally active in its native form (mean ± SEM of relative luciferase activity of promoter-less vector vs. IL-15 promoter vector = 77.00 ± 3.29 vs. 626.8 ± 25.05 respectively, N=3 each, P<0.0001).  Deletion of the putative Zeb1 binding sites (BS#1, BS#2, BS#3 and BS#1-3) or the entire Zeb-binding region (BR) in the IL-15 promoter led to a significant increase in IL-15 transcription as determined by relative luciferase activity (mean ± SEM of relative luciferase activity of pGL3 IL-15 vs. BS#1 vs. BS#2 vs. BS#3 vs. BS#1-3 vs. BR vectors = 99.56 ± 0.33 vs. 7135 ± 108.5 vs. 3940 ± 62.36 vs. 2372 ± 24.65 vs. 1099 ± 9.72 vs. 1525 ± 32.00, n=4 each; P<0.0001 each). Since CpG methylation of DNA can physically prevent transcription factor binding to regulatory regions of DNA, we hypothesized that CD4+ cells from CTCL patients might display reduced binding of Zeb1 to the IL-15 promoter thereby increasing IL-15 transcription. Indeed, using chromatin immunoprecipitation (ChIP)-PCR, we observed a substantial loss of Zeb1 binding at the IL-15 promoter in the CD4+ cells of CTCL patients compared to those of normal donors (mean ± SEM of relative Zeb1 binding to IL-15 promoter in normal donor vs. CTCL patient CD4+ T-cells = 101.3 ± 1.48 vs. 25.31 ± 3.78, n=3 each; P<0.0001). Similarly, silencing Zeb1 in normal donor CD4+ T-cells caused an increase in IL-15 transcription. Finally, constitutive IL-15 overexpression was sufficient to cause a high penetrance lymphoproliferative disease in transgenic mice, with typical dermatologic features of human CTCL, including alopecia, scaly erythematous plaques/patches, and ulcerations, caused by an epidermotropic T-cell infiltrate. Thus, we defined Zeb1 as a novel repressor for IL-15 transcription in normal T-cells and show that epigenetic interference with its repressive function at the IL-15 promoter is associated with T-cell lymphoma in vivo, highlighting the role of epigenetic processes in inflammation-induced cancers and further supporting the role of Zeb1 as a putative tumor suppressor gene in CTCL