Cutaneous T-cell lymphoma (CTCL) is the most common type of primary cutaneous lymphoma. Here we report that CTCL patients show increased interleukin-15 (IL-15) in a clinical stage-dependent manner. Mechanistically, we show that Zeb1 is a transcriptional repressor of IL-15 in T-cells and that hypermethylation of the Zeb1 binding region within the IL-15 promoter, as seen in CTCL patients, prevents Zeb1 binding and causes increased transcription of IL-15. Using a transgenic mouse model of IL-15, we provide evidence that overexpression of IL-15 induces a spontaneous CTCL that mimics the human neoplasm. Excessive autocrine production of IL-15 in T-cells inhibits an HDAC1-mediated negative autoregulatory loop, resulting in the upregulation of HDAC1 and HDAC6, and transcriptional induction of the onco-miR-21. Interruption of IL-15 downstream signaling with isotype-specific HDAC inhibitors halts (HDAC1) or significantly delays (HDAC6) the progression of CTCL in vivo and provides pre-clinical evidence supporting a hierarchical model of oncogenic signaling in CTCL.

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 Disclosures Porcu: Cell Medica: Research Funding; Celgene: Research Funding; Shape: Research Funding; Infinity: Research Funding; Seattle Genetics: Research Funding.

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<p>Supplementary Table 1A (ST1A). Clinical features of CTCL (MF and SS) patients used in Figure 1B and Figure 4E (denoted with *). Stage 1B and IIB are MF samples and Stage III/IV are SS samples. Supplementary Table 1B (ST1B). Clinical features of SS patients whose CD4+ T cells were used in Figure 1C. Supplementary Table 1C (ST1C). Clinical features of SS patients whose CD4+ T cells were used in Figure 5B and Supplementary Figure 1D-F (denoted with #). Supplementary Table 1D (ST1D). Clinical features of SS patients whose CD4+ T cells were used in Figure 4D and 5A.</p>

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<p>Supplementary Figures 1 - 7. Supplementary Figure 1. Immunoblot analysis of IL-15 in normal donor CD4+ T-cells, SS patient CD4+ T-cells and patient derived cell lines. Supplementary Figure 2. Representative flow cytometric histogram is shown for CD3 expression in mononuclear cells from skin of WT and IL-15 tg mice. Supplementary Figure 3. Immunoblot analysis of HDAC1 in normal donor CD4+ T-cells, SS patient CD4+ T-cells and patient derived cell line, Hut78. Protein lysates were probed for expression of HDAC1; β-Actin was used as housekeeping control. Supplementary Figure 4. Immunoblot analysis for expression of p21 protein in normal donor CD4+ cells following ten days of incubation in IL-15 compared to unstimulated normal donor CD4+ T-cells. Supplementary Figure 5. Pie chart showing the distribution of HDAC1 occupancy in normal donor CD4+ T-cells (upper left) compared to patient CD4+ T-cells (upper right) and normal donor CD4+ T-cells stimulated with 100ng/ml IL-15 for 24 hours (lower left). Supplementary Figure 6. High magnification and (B) low magnification histological analysis of IL-15 Tg skin. Supplementary Figure 7. The CTCL mouse disease severity scoring system is on a 0 to 100 scale, with 100 being the highest (most severe) score.</p>