Cutaneous T-cell lymphoma (CTCL) is the most common type of primary cutaneous lymphoma. Here, we report that patients with CTCL show increased IL15 in a clinical stage–dependent manner. Mechanistically, we show that ZEB1 is a transcriptional repressor of IL15 in T cells and that hypermethylation of the ZEB1 binding region within the IL15 promoter, as seen in patients with CTCL, prevents ZEB1 binding and causes increased transcription of IL15. Using a transgenic mouse model of IL15, we provide evidence that overexpression of IL15 induces a spontaneous CTCL that mimics the human neoplasm. Excessive autocrine production of IL15 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 IL15 downstream signaling with isotype-specific HDAC inhibitors halts (HDAC1) or significantly delays (HDAC6) the progression of CTCL in vivo and provides preclinical evidence supporting a hierarchical model of oncogenic signaling in CTCL.

Significance: To date, CTCL pathogenesis remains unknown, and there are no curative therapies. Our findings not only demonstrate a critical role for IL15-mediated inflammation in cutaneous T-cell lymphomagenesis, but also uncover a new oncogenic regulatory loop in CTCL involving IL15, HDAC1, HDAC6, and miR-21 that shows differential sensitivity to isotype-specific HDAC inhibitors. Cancer Discov; 6(9); 986–1005. ©2016 AACR.

This article is highlighted in the In This Issue feature, p. 932

Among primary cutaneous lymphomas, cutaneous T-cell lymphoma (CTCL) is by far the most common type (1). The majority of CTCL consists of two related CD4+ mature T-cell neoplasms: Mycosis fungoides (MF) and Sezary syndrome (SS). MF originates from a clonal expansion of skin-homing CD4+ memory T cells, usually presents with limited skin involvement, and is initially characterized by an indolent clinical course with stepwise progression toward greater tumor burden in the skin, followed in a subset of patients by extracutaneous dissemination. SS is a more aggressive type of CTCL that can present de novo or as an advanced-stage progression of MF and is characterized by erythroderma, lymphadenopathy, and circulating clonal atypical CD4+ T cells. Survival in MF/SS varies according to clinical stage, which is defined according to a composite tumor, node, metastasis, and blood (TNMB) classification, as revised by the European Organisation for Research and Treatment of Cancer/International Society for Cutaneous Lymphomas (2). Patients with “early-stage” disease have skin-limited involvement with superficial patches or plaques and an expected survival of >10 years. Patients with “advanced-stage” disease, which includes all patients with SS, have skin tumors, erythroderma, and/or extracutaneous involvement, and median survival < 5 years. Although discrete but highly overlapping molecular signatures associated with MF and SS have been described, the cancer-initiating events and the oncogenic drivers in CTCL remain largely unknown, and there are no curative therapies. Several pan-inhibitors of histone deacetylase (HDAC) have been approved for treatment of CTCL, yet none are curative and each is associated with significant toxicity (3, 4). In the experimental setting, the term CTCL has been used synonymously with MF/SS.

Cytokines can affect T-cell proliferation and survival during various stages of T-cell development and homeostasis. In CTCL, cytokines such as IL4, IL7, IL13, IL15, IL16, IL17, and IL31 are present and/or dysregulated during various stages of disease progression (5–10). Because IL15 was first proposed to be essential for lymphoma cell growth in vitro, numerous studies have confirmed the pathogenic role of IL15 in T-cell lymphoma (5, 6, 11–14). IL15 is a pleiotropic cytokine that utilizes a nontransducing, private α-chain and a common IL2/IL15 βγ-signal transducing complex to function as a growth and survival factor for T cells and natural killer cells (15, 16). There is evidence suggesting that IL15 has a pathogenic role in CTCL. IL15 is highly stimulatory for CD4+ CTCL cells in vitro (10, 17–19), and strong IL15 expression in lesional skin is a characteristic feature of CTCL because normal skin does not express significant amounts of this cytokine, and the reported prevalence and intensity of IL15 expression in inflammatory skin disorders such as atopic dermatitis and psoriasis are variable (14, 20). In CTCL, IL15 has been implicated in the recruitment of CD4+ memory T cells to the skin, induction of T-cell proliferation, and inhibition of apoptotic cell death (17, 21). Human CD4+ CTCL cells produce IL15 in culture, thereby sustaining cell growth in an autocrine fashion (10). An increase in autocrine IL15 production during disease progression has been proposed to promote CTCL cell independence from the skin microenvironment and the development of extracutaneous disease (14). Furthermore, IL15 can induce skin-homing CD4+ memory cells to acquire a regulatory T-cell phenotype in vitro, which may protect the malignant cells from elimination through immune evasion (22). Additional data have established that hair follicle–derived IL15 and IL7 regulate epidermotropism of memory T cells in inflammatory skin diseases and malignant lymphoma (23).

Although the malignant CD4+ T cells from blood of all patients with SS we have tested appear to produce and be activated by IL15, the role of this cytokine in the pathogenesis of CTCL remains unresolved. Here, we show that overexpression of IL15 in patients with CTCL is coincident with epigenetic disruption of ZEB1 binding at the IL15 promoter. In vitro, disruption of ZEB1 binding results in overexpression of IL15, which in turn drives HDAC1 and HDAC6 upregulation and activation of onco-miR-21. In vivo, overexpression of IL15 in a mouse model recapitulates human CTCL, whereas pharmacologic inhibition of HDAC1 and HDAC6 prevents the progression of CTCL.

CD4+ T Cells from Patients with CTCL Display IL15 Promoter Methylation in the ZEB1 Repressor Binding Region

To study the expression of IL15 in CTCL, lesional skin biopsies and peripheral blood lymphocytes from patients with CTCL were analyzed for IL15 expression. IL15 protein is strongly expressed in CTCL, with intense and specific positive staining of atypical lymphoid cells in Pautrier's microabscesses that are pathognomonic for CTCL (Fig. 1A), whereas normal human skin and T cells do not express IL15 (Supplementary Fig. S1A; ref. 17). We then measured expression of IL15 mRNA in highly enriched peripheral blood CD4+ T cells (>90% pure as determined by flow cytometry) from patients with CTCL in various stages of disease. Relative to normal donor CD4+ cells, peripheral blood CD4+ cells from patients with CTCL listed in Supplementary Table S1A expressed significantly more IL15 transcript, which was proportional to disease severity in patients (mean ± SEM of fold increase in Stage IB vs. Stage IIB vs. Stage III/IV = 2.58 ± 0.70 vs. 5.38 ± 2.09 vs. 7.47 ± 1.78; n = 3 each; P = 0.032, 0.005, and 0.011, respectively; Fig. 1B).

Because epigenetic modifications such as DNA methylation can alter gene expression, we selected samples highly enriched in neoplastic T cells from the blood of patients with SS listed in Supplementary Table S1B and asked if promoter methylation of the IL15 gene might influence its transcriptional regulation. We observed significantly higher overall CpG methylation within the IL15 promoter in CD4+ T cells from patients with SS versus normal donors (mean ± SEM of relative % methylation = 232.0 ± 49.18, n = 9 vs. 100.1 ± 7.619, n = 6; P = 0.03; Fig. 1C). Furthermore, methylation at the IL15 promoter was higher in malignant CD4+ T cells compared with nonmalignant 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; Fig. 1C). Using the same approach, we analyzed methylation of each “CpG dinucleotide” in the IL15 promoter (Fig. 1D). Once again, methylation was higher in CD4+ T cells from patients with CTCL than CD4+ T cells from normal donors and, within each patient, in CD4+ T cells versus neutrophils (Fig. 1E and Supplementary Fig. S1B). This CpG-rich region of the human IL15 promoter contains three putative binding sites for the known transcriptional repressor ZEB1 (Fig. 1D and Supplementary Fig. S1C; refs. 24, 25).

In order to examine the effect of CpG methylation (at the ZEB1 binding region) on IL15 transcription, the CpG-rich 5′ regulatory region of the IL15 promoter (0.5 kb upstream of transcription start site) was amplified and cloned into PGL3 luciferase vector. As measured by relative luciferase activity, this region of the IL15 promoter is transcriptionally active in its native form (data not shown). The deletion of the putative ZEB1 binding sites (BS#1, BS#2, BS#3, and BS#1–3) or the entire binding region in the IL15 promoter led to a significant increase in IL15 transcription as determined by relative luciferase activity (mean ± SEM of relative luciferase activity of pGL3 IL15 vs. BS#1 vs. BS#2 vs. BS#3 vs. BS#1–3 vs. BR vectors = 99.56 ± 0.33 vs. 7,135 ± 108.5 vs. 3,940 ± 62.36 vs. 2,372 ± 24.65 vs. 1,099 ± 9.72 vs. 1,525 ± 32.00, n = 4 each; P < 0.0001 each; Fig. 1F). We suspect that the deletion of the entire binding region is less effective than deleting BS#1–3 because the former likely includes binding sites for transcriptional activators in the regulatory region of the IL15 gene.

Because CpG methylation of DNA can physically prevent transcription factor binding to a regulatory region of DNA, we hypothesized that CD4+ cells from patients with CTCL might display reduced binding of ZEB1 to the IL15 promoter, thereby increasing IL15 transcription. Indeed, using chromatin immunoprecipitation (ChIP)–PCR, we observed substantial loss of ZEB1 binding at the IL15 promoter in CTCL patient CD4+ cells compared with normal donor CD4+ cells (mean ± SEM of relative ZEB1 binding to IL15 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; Supplementary Fig. S1D; and Supplementary Table S1C). Of note, the expression levels of ZEB1 binding protein were similar in patients with SS, normal donors, and patient-derived CTCL cell lines (Supplementary Fig. S1E and S1F). In addition, silencing ZEB1 in normal donor CD4+ T cells caused increase in IL15 transcript (Supplementary Fig. S1G). These experiments show that CpG methylation within the ZEB1 binding sites of the IL15 promoter blocks ZEB1 binding and increases IL15 expression. This suggests that overexpression of IL15 in CTCL patient samples is due, at least in part, to this epigenetic event.

IL15 Overexpression in the Mouse Mimics Human CTCL

We confirmed that IL15 is expressed by the neoplastic T cells in lesional skin and that IL15 expression is increased in a stage-dependent manner in purified peripheral blood CD4+ T cells from patients with CTCL, compared with normal donors. Next, we proceeded to elucidate the role of IL15 in the development of CTCL using Il15 transgenic (tg) mice. We had earlier reported that approximately 30% of the Il15 tg colony rapidly succumbed to an aggressive leukemia of large granular lymphocytes (LGL; refs. 26, 27). We subsequently observed that the remaining 70% of IL15 tg mice developed progressive alopecia and extensive cutaneous lesions within 4 to 6 weeks of birth. The dermatologic features of the Il15 tg mice display many of those observed in human CTCL, including full-body, scaly erythematous plaques/patches, exfoliative dermatitis, ulcerations (Fig. 2A), and severe pruritus. The mice that develop the CTCL phenotype display a significantly longer survival compared with those with the aggressive LGL leukemia, but still have a significantly shorter life span compared with wild type (WT) littermate controls (median age of Il15 tg vs. WT = 50.4 weeks vs. 80 weeks, P < 0.0003; Fig. 2B). Il15 tg CTCL mice have only moderate increases in peripheral blood white blood cell (WBC) counts compared with age-matched WT controls or leukemic mice (mean ± SEM of WBC count in WT vs. leukemic Il15 tg vs. CTCL Il15 tg = 7,225 ± 1,211 vs. 140,750 ± 25,103 vs. 32,609 ± 4,483; n = 4, 10, and 11, respectively; Fig. 2C). Although extreme leukocytosis is not a feature of the Il15 tg CTCL mice, peripheral blood smears show the presence of atypical lymphocytes with hyperchromatic nuclei, with folded and indented nuclear membrane, which is one of the hematologic manifestations of late-stage patients with MF and patients with SS (Fig. 2D).

To compare the pathology and immunophenotype of the spontaneous CTCL in Il15 tg mice and human CTCL, we performed a histologic analysis of the skin. Human CTCL can be distinguished from many inflammatory skin disorders by the presence of Pautrier's microabscesses in the epidermis, which reflect the epidermotropic nature of the lymphoid infiltrate and, when found, are a hallmark of the disease (28). Histologic analysis of lesional skin from Il15 tg mice revealed marked infiltration of the dermal–epidermal junction with atypical lymphocytes, with cells tracking to the upper skin layers and formation of classic Pautrier's microabscesses (Fig. 3A, compare with 1A). Like human CTCL, the cutaneous lymphoid infiltrate of Il15 tg mice predominantly consisted of both CD3+CD4+ and CD3+CD8+ T cells, with an approximately 25-fold increase in CD3+ T cells compared with WT littermate controls (mean ± SEM of absolute cell number in WT vs. Il15 tg skin = 0.15 ± 0.09 vs. 3.80 ± 1.86; n = 8 and 14, respectively; P = 0.0001; Fig. 3B; Supplementary Fig. S2A and S2B). The cutaneous lymphoid infiltrate in the Il15 tg mice showed robust expression of the skin-homing molecules cutaneous lymphocyte antigen (CLA) and chemokine receptor 4 (CCR4; Supplementary Fig. S2C). In addition, skin CD4+ T cells from the Il15 tg mice showed significantly higher expression of the adhesion molecule CD44 (mean ± SEM of CD4+CD44+ expression in WT vs. Il15 tg skin = 12.28 ± 4.35 vs. 34.99 ± 2.44; n = 8 and 14, respectively; P < 0.0001), as observed in human CTCL cells (Supplementary Fig. S2D; refs. 29, 30). Likewise, CD62L expression was either lost or reduced in the CD4+ T cells of Il15 tg mice, compared with WT mice (mean ± SEM of CD4+CD62L expression in WT vs. IL15 tg skin = 70.67 ± 4.74 vs. 88.56 ± 2.23; n = 8 and 14, respectively; P = 0.0009; Supplementary Fig. S2E; ref. 31). T-cell receptor (TCR)–Vβ staining of T cells isolated from the skin of IL15 tg mice revealed the simultaneous expansion of select clonal populations of T cells, none sufficiently large to emerge as a dominant clone, resulting in an oligoclonal pattern (Supplementary Fig. S2F). In addition, CD26 expression was reduced in skin of Il15 tg mice compared with WT mice (mean ± SEM of CD26 expression in WT vs. Il15 tg skin = 33.77 ± 1.235 vs. 6 ± 0.611; n = 3 each; P < 0.0001; Supplementary Fig. S2G). Furthermore, skin resident mononuclear cells overexpressed PLS3, GATA3, and CD164 in Il15 tg mice compared with WT mice (Supplementary Fig. S2H).

The validity and impact of a spontaneous murine cancer model are enhanced by the demonstration that the malignant cells are able to engraft, expand, and mimic the primary disease in secondary recipient mice (32). To test this, we transplanted mononuclear cells isolated from the skin of Il15 tg mice (approximately 35 weeks old) into recipient SCID mice, as shown in the experimental schema (Fig. 3C). Because the heavy CD3+ cutaneous T-cell infiltrate in the CTCL Il15 tg mice is a mixture of CD4+ and CD8+ lymphocytes, often with a predominance of CD8+ cells, unsorted cells were transplanted subcutaneously to determine which subset displayed the neoplastic phenotype (Fig. 3D, left). We observed two patterns of transplantable CTCL growth in secondary mice. In a subset of secondary recipient mice, 6 weeks after transplantation, discrete tumor-like lesions were observed throughout the dorsal skin (Fig. 3D, right), caused by cutaneous infiltration with atypical CD3+CD4+CD8 T cells spanning the dermis and epidermis (Fig. 3E–H). In the same mice, an approximately 12-fold expansion of CD4+ T cells and a near- complete absence of CD8+ T cells were observed in the peripheral blood. These observations identify the CD4+ T-cell subset as the malignant population in the primary Il15 tg mouse. In another subset of transplanted mice, the development of secondary skin lesions took longer to evolve (∼14 weeks) but resulted in a more aggressive form of secondary CTCL (Fig. 3I) with a florid leukemic component due to the expansion of a double-negative CD3+CD4CD8 T-cell population that was clonal for TCR-Vβ7 and consisted of morphologically atypical lymphocytes with cerebriform hyperchromatic nuclei similar to classic SS cells (Fig. 3J). Hematoxylin and eosin (H&E) staining of the skin lesion in this subset of mice also revealed a deep patchy infiltrate of large lymphoid cells, with a residual epidermotropic component, as shown by the formation of Pautrier-like structures (Fig. 3K). Similar to the peripheral blood, the cutaneous lymphoid infiltrate was predominantly CD3+CD4CD8 (Fig. 3L and M; Supplementary Fig. S2I). Thus, in spite of the variable immunophenotype, the transplantation of skin mononuclear cells from primary CTCL mice resulted in a range of secondary T-cell neoplasms that again reflected the clinical and histologic spectrum of human CTCL (33). Notably, secondary recipient mice transplanted with splenic mononuclear cells from Il15 tg mice did not develop skin lesions or secondary T-cell lymphoma, suggesting that the malignant cells from the primary CTCL mice are highly enriched in or limited to the skin in some cases (data not shown). Further, these findings support the notion that the neoplastic T-cell population within the skin of the Il15 tg mice consists of the CD3+CD4+CD8 and CD3+CD4CD8 T cells. In addition, malignant cells from secondary mice overexpressed IL15Rα and displayed significantly enhanced cell death in the presence of the JAK1/3 inhibitor tofacitinib, suggesting that IL15 signaling is a requisite survival pathway in these cells (Supplementary Fig. S2J and S2K).

IL15 Regulates HDAC Expression

Patients with CTCL show clinical responsiveness to pan-HDAC inhibitors (HDACi); however, insight as to the mechanisms of HDAC expression in CTCL is limited (34). Along with the elevated expression of IL15 in CTCL patient samples, we observed that patients' peripheral blood CD4+ T cells have increased expression of HDAC1 and HDAC6 transcripts through different stages of the disease when compared with normal donors (Fig. 4A). Although not statistically significant, there was a trend for higher expression of HDAC1 and HDAC6 in more advanced stages of CTCL. Similar increases in protein levels of HDAC1 were observed in blood CD4+ cells from patients with SS as well as in the SS patient–derived cell line Hut78, compared with normal donors (Supplementary Fig. S3A and S3B). In order to test the hypothesis that IL15 modulates HDAC expression, we exposed normal donor CD4+ T cells to IL15 in vitro and measured HDAC protein levels. We observed upregulated levels of HDAC1 and HDAC6 in normal CD4+ T cells after 48 hours of exposure to IL15 in vitro (Fig. 4B and C). We then examined the intracellular distribution of HDAC1 and HDAC6 before and after exposing normal CD4+ T cells to IL15. We observed that although HDAC1 was nuclear regardless of IL15 exposure, HDAC6 (that was both cytoplasmic and nuclear before IL15 incubation) translocated exclusively to the cytoplasm upon incubation with IL15 (Supplementary Fig. S3C and S3D). The IL15-mediated induction of HDACs was not associated with increased CD4+ T-cell growth (Supplementary Fig. S3E).

In order to determine the mechanism(s) of HDAC1 and HDAC6 induction by IL15, we first investigated a previously described negative autoregulatory feedback loop where HDAC1 binds directly to its own promoter and represses transcription (35). Using antibodies specific for HDAC1, we probed the interaction of HDAC1 with its own promoter in CTCL CD4+ T cells, resting normal donor CD4+ T cells, and IL15-stimulated normal donor CD4+ T cells by ChIP-seq, which combines ChIP with sequencing of DNA associated with the protein of interest, i.e., HDAC1. ChIP-seq data showed that CTCL patient CD4+ T cells displayed decreased occupancy of HDAC1 at its own promoter when compared with resting normal donor CD4+ T cells (Supplementary Table S1D and Fig. 4D and E). We also observed reduced binding of HDAC1 to its promoter in IL15-stimulated normal CD4+ T cells when compared with resting normal donor CD4+ T cells (Fig. 4D and E), suggesting that the overexpression of HDAC1 seen in CTCL patient CD4+ T cells is due, at least in part, to an IL15-induced decrease in HDAC1 occupancy at the HDAC1 promoter. In order to investigate HDAC1′s role as a transcriptional repressor for HDAC1 and HDAC6, normal donor T cells were transfected with shHDAC1 and evaluated for changes in HDAC1 and HDAC6 transcripts by RT-PCR. Silencing of HDAC1 in resting normal donor CD4+ cells leads to increased transcription of both HDAC1 and HDAC6 mRNAs, suggesting HDAC1 is a transcriptional repressor of both HDAC1 and HDAC6 in CTCL patient CD4+ T cells and in normal donor CD4+ T cells upon IL15 stimulation (Fig. 4F). Thus, decreased binding of HDAC1 to its own promoter and to the HDAC6 promoter in IL15-expressing CTCL CD4+ T cells in vivo, and in normal CD4+ T cells following stimulation with IL15 in vitro, results in their increased expression, whereas knockdown of HDAC1 results in upregulation of both HDAC1 and HDAC6 in normal resting CD4+ T cells. These data confirm that HDAC1 is a physiologic transcriptional repressor of HDAC1 (35) and show, for the first time, that it is also a physiologic transcriptional repressor of HDAC6. Further, we show that chronic stimulation of CTCL CD4+ T cells by IL15 disrupts the negative autoregulatory loop of HDAC1, thereby contributing to the upregulation of HDAC1 and HDAC6 in vivo (Fig. 4G).

Given the broad effects of HDACs on the signaling pathways involved in cancer cell growth and survival (36), we measured the downstream effects of IL15-induced increase in HDAC expression in normal CD4+ T cells. For example, we confirmed overexpression of HDAC1 in normal donor CD4+ T cells stimulated with IL15, and the consequent silencing of its downstream target p21 (Supplementary Fig. S4A; ref. 37). Because lymphocyte migration is regulated by HDAC6 (38) and important in CTCL, we showed that knockdown of HDAC6 by shRNA in normal donor CD4+ T cells reduced cell migration toward IL15, when compared with controls (mean ± SEM of T-cell migration in shGFP control vs. shHDAC6 Clone #3 vs. shHDAC6 Clone #5 = 49.67 ± 3.28 vs. 20.00 ± 2.88 vs. 26.67 ± 1.66; n = 3 each; P = 0.0025 and 0.0033, respectively; Supplementary Fig. S4B). Knockdown of HDAC1 did not affect T-cell migration, suggesting that HDAC1 does not influence IL15-induced migration of T cells (Supplementary Fig. S4B). These observations are consistent with the notion that the IL15–HDAC1/6 axis contributes to the pathogenesis of CTCL through multiple mechanisms.

Genome-Wide Mapping of HDAC1 in CTCL

Although HDAC1 is overexpressed in CTCL, the relationship of its overexpression to gene regulation has not been clearly defined. Here, we interrogated the epigenetic landscape of HDAC1 in highly enriched malignant CD4+ T cells from a patient with SS and from normal donor CD4+ T cells (the latter with or without IL15 stimulation). We found that there are parallels in HDAC1 occupancy between CD4+ T cells from a patient with SS and from normal donor CD4+ T cells stimulated with IL15. Overall HDAC1 occupancy at intron and 5′-UTR regions were similar among these two cell populations when compared with resting normal donor CD4+ T cells, whereas no changes in HDAC1 occupancy were noted among these three CD4+ T-cell populations in seven other genomic locations that were analyzed (i.e., distal promoter, proximal promoter, exon, 3′-UTR, proximal downstream, distal downstream, and distal intergenic regions). Specifically, CD4+ T cells from the patient with SS and from the normal donor CD4+ T cells exposed to IL15 showed decreased HDAC1 occupancy at the intron location and increased overall genomic HDAC1 occupancy at the 5′-UTR locations compared with resting normal donor CD4+ T cells (Supplementary Fig. S5A). To determine whether HDAC1 binding patterns were indicative of a broader trend in transcriptional regulation, we examined the relationship between HDAC1-enriched sites with enhancer mark H3K27Ac to measure levels of gene expression on a global scale (39). As shown, the “Promoter Plots” show the tag distribution across all human RefSeq promoters (25,143 regions), the “Active Regions Plots” show the tag distribution at all H3K27Ac/HDAC1 peak locations (35,725 regions), and the “Super Enhancers (SE) Plots” show the tag distribution at all H3K27Ac and HDAC1 SE locations (1,525 regions). Consistent with HDAC1′s known role as a repressor of gene transcription, our data suggest that occupancy patterns of HDAC1 (resting normal donor CD4+ T cells > normal donor CD4+ T cells stimulated with IL15 > SS patient CD4+ T cells) are inversely proportional to histone H3K27Ac (CTCL patient CD4+ T cells > normal donor CD4+ T cells stimulated with IL15 > normal donor CD4+ T cells), which is a mark for active enhancers (Supplementary Fig. S5B; ref. 39). Although there was significant overlap in occupancy between the three types of samples, unique patterns were observed (Supplementary Fig. S5C). Using Ingenuity Pathway Analysis (IPA; ref. 40) to functionally annotate HDAC1 target genes, we identified the top biological processes regulated by HDAC1 in normal (with or without IL15) CD4+ T cells and in CD4+ T cells from the patient with SS (Supplementary Fig. S5D). IPA identified super clusters of high-scoring functions in each of the three data sets, revealing dominance of a gene expression network in IL15-stimulated normal donor CD4+ T cells and in CD4+ T cells from the patient with SS (Supplementary Fig. S5D, orange and green panels, respectively). Interestingly, cell cycle is the most enriched pathway in normal resting CD4+ T cells, suggesting a role for HDAC1 in maintaining the physiologic cell-cycle gene network (Supplementary Fig. S5D, blue panel). Importantly, although the comparison of genome-wide HDAC1 landscape and consequent pathway analysis in CD4+ cells from the SS patient, normal donor, and IL15-stimulated normal donor is revealing, our very small sample size (n = 1 each) limits our interpretation of these data.

HDAC1 Is a Positive Regulator of Onco-miR miR-21

We have previously shown the involvement of HDAC1 in the regulation of miRNA expression (27). Although a considerably large proportion of HDAC1 occupancy was observed in the resting normal donor CD4+ T-cell genome, we identified a few gene loci where a higher proportion of HDAC1 occupancy was observed in the SS patient CD4+ T cells and in IL15-stimulated normal donor CD4+ T cells (data not shown). One such region of interest was the promoter of the miR-21, a bona fide “onco-miR” (41) that is overexpressed in patients with CTCL (Fig. 5A; Supplementary Fig. S5E; Supplementary Table S1D; refs. 42–44). Consistent with this observation, we found significantly increased levels of miR-21 in the SS patient CD4+ T cells and in IL15-stimulated normal donor CD4+ T cells compared with resting normal donor CD4+ T cells (Fig. 5B and Supplementary Table S1C). Circulating CD4+ T cells in patients with CTCL with MF also exhibited increased levels of miR-21 compared with normal donor cells (Supplementary Fig. S5F and Supplementary Table S1C). Interestingly, similar observations were made when comparing circulating normal CD4+ T cells in WT with Il15 tg mice with early-stage disease. Circulating CD4+ cells (from peripheral blood) as well as skin resident cells in Il15 tg mice overexpressed miR-21 in comparison with WT counterparts (Supplementary Fig. S5G). To test if the occupancy of HDAC1 on miR-21 promoter affects its transcription, we silenced HDAC1 in the CTCL cell line Hut78 using shRNA directed against HDAC1 and showed downregulation of miR-21 (Fig. 5C). Similarly, treatment of Hut78 cells with an HDAC1/2 inhibitor (Merck60) caused decreased expression of miR-21 (Fig. 5D). Therefore, along with the known role of HDAC1 as transcriptional repressor, we determined that HDAC1 is a positive regulator of mir-21 both in CTCL patient CD4+ T cells and in IL15-stimulated normal donor CD4+ T cells.

Inhibition of HDAC Activity Prevents Progression of CTCL In Vivo

Pan-HDAC inhibitors show strong antitumor activity in a subset of patients with CTCL (45). Considering that overexpression of IL15 leads to increased HDAC1 and HDAC6 in our preclinical model, we used specific HDACis to target HDAC1/2 (Merck60; ref. 46) and HDAC6 (WT161; ref. 47) either individually or in combination. Hut78 cells were more sensitive to cell death with the HDAC1/2-specific inhibitor than with the HDAC6 inhibitor or with the combination (mean ± SEM of relative viability in Hut78 control vs. Merck60 vs. WT161 vs. combination treatment = 100.0 ± 16.36 vs. 44.65 ± 2.97 vs. 60.94 ± 3.24 vs. 40.32 ± 0.90, n = 4 each, P < 0.0001; Fig. 6A). To test the relative in vivo efficacy of these inhibitors, 4-week-old IL15 tg mice with early evidence of CTCL (Supplementary Fig. S6A–S6D) were treated with 50 mg/kg of either the HDAC1/2-specific inhibitor or the HDAC6-specific inhibitor or the combination, 5 days/week for 4 weeks (Fig. 6B). As indicated by our CTCL severity score (Supplementary Fig. S7; http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3447447/), placebo-treated Il15 tg control mice progressively developed the typical cutaneous lesions, whereas the HDAC1/2-specific inhibitor Merck60 alone halted and even regressed clinical progression of CTCL in the Il15 tg mice [mean ± SEM of relative CTCL severity (Supplementary Fig. S7A–S7E) in Placebo vs. Merck60 vs. WT161 vs. combination treatment groups, P = 0.038; Fig. 6C–E]. IL15 tg mice treated with the HDAC6-specific inhibitor WT161 showed delay in progression of CTCL compared with placebo-treated mice, but WT161 was less effective than Merck60 (Fig. 6C–E). These observations were further corroborated by histopathologic analysis of skin sections from treated mice, as Il15 tg mice treated with Merck60 had the fewest CD3+CD4+ infiltrates in the skin compared with the other treatment groups (Fig. 6D). Taken together, our preclinical data show that targeting HDAC1/2 with Merck60 effectively inhibits progression of early CTCL in the Il15 tg mice and support a similar approach in the treatment of human CTCL with the hope of reducing toxicities associated with the use of pan-HDACis (Fig. 6E).

IL15 has been previously implicated in T-cell lymphoproliferative disorders (48, 49), and overexpression of IL15 in clinical samples obtained from patients with CTCL has been documented by several groups (10, 14, 17). However, why IL15 expression is increased in CTCL and what the potential role of this cytokine is in the initiation and progression of CTCL remain poorly understood. With the intent of gaining insight into mechanisms of IL15 overexpression and its role in CTCL pathogenesis, we first confirmed the relevance of IL15 by showing stage-dependent overexpression in lesional skin and peripheral blood CD4+ T cells from patients with CTCL. We elucidated at least one mechanism by which IL15 can be overexpressed in vivo, showed how this cytokine induces specific HDACs in CTCL CD4+ T cells, and then investigated the biological and clinical aspects of a spontaneous epidermotropic CTCL that develops in mice that constitutively overexpress IL15. We demonstrated that the CTCL in Il15 tg mice closely resembles human CTCL, based on clinical, histopathologic, and immunophenotypical criteria. We also found shared molecular aberrations between the CTCL in Il15 tg mice and human CTCL, which could be replicated in normal human CD4+ T cells exposed to IL15 in vitro. Finally, we interrogated the animal model with the purpose of identifying mechanisms and pathways amenable to selective pharmacologic targeting for preclinical validation, demonstrating some success at halting the progression and inducing the regression of CTCL in vivo using an HDAC1-specific inhibitor (Fig. 7).

Mechanism of IL15 Overexpression in CTCL

Although the Il15 tg mouse provides a novel and informative experimental platform to study the oncogenic effects of IL15 in CTCL and explore new treatments, it cannot by itself offer insight into the mechanisms of IL15 overexpression in patients. Therefore, we examined the structure of the IL15 gene in patients with CTCL and studied the epigenetic regulation of its transcription. Mutations, insertions, and deletion of the transcriptional checkpoints in the IL15 gene have previously been implicated in the constitutive overexpression of this cytokine in T-cell lymphoma cell lines, suggesting that structural alterations of the gene can modulate IL15 production (50–52). Although we did not find any genetic alterations in the IL15 promoter isolated from CTCL patient CD4+ T cells (data not shown), we did observe an epigenetic alteration (i.e., hypermethylation) at the IL15 promoter in these cells when compared with normal donor CD4+ T cells. Hypermethylation of CpG islands in the 5′ regulatory region of genes can alter gene expression through mechanisms that do not involve changes in nucleotide sequence (53, 54). Genes such as BCL7a, PTPRG, THBS4, MGMT, p73, p16, p15, CHFR, SHP1, and TMS1 display widespread promoter hypermethylation in CTCL patient CD4+ T cells (55, 56). In addition to gene silencing, hypermethylation can also result in gene activation by inhibiting access of transcriptional repressors to promoter regulatory regions (57, 58). Hypermethylation of repressive sequences in the hTERT gene has been shown to upregulate hTERT expression in high-risk human papillomavirus (HPV)–induced cervical cancer (59). Our data show that hypermethylation of CpG islands in the IL15 promoter within CTCL patient CD4+ T cells prevents the transcriptional repressor ZEB1 (also known as TCF8) from binding and negatively regulating IL15 expression, thus providing an explanation for the overexpression of IL15 in CTCL patient CD4+ T cells and offering the first evidence for the epigenetic regulation of its expression. This role is also consistent with other well-characterized functions for ZEB1 in T cells, such as rapid shut-off of IL2 transcription after antigen recognition and negative regulation of Class I MHC-restricted T cell–associated molecule (CRTAM; ref. 60). The fact that loss of genetic material at 10p11.2, where the ZEB1 gene is located, as well as loss-of-function somatic mutations, has been observed in a subset of patients with CTCL further supports a tumor-suppressor role for ZEB1 in CTCL, highlighting multiple alternative mechanisms for the loss of negative IL15 regulation (61–63). The patients tested in our study were positive for ZEB1 protein, indicating that genetic deletions of ZEB1 could be ruled out as a causative mechanism for IL15 overexpression. Finally, loss of ZEB1 has also been observed in primary cells from another type of mature T-cell neoplasm, adult T-cell leukemia (ATL), and genetic disruption of ZEB1 in mice results in the development of CD4+ T-cell lymphomas with a 100% penetrance (64). Interestingly, overexpression of another gene, HMGA1, in mice induces upregulation of IL15, leading to the development of T-cell lymphomas (65). These results, together with our finding that knockdown of ZEB1 causes upregulation of IL15 expression in resting normal human CD4+ T cells, suggested that the binding of ZEB1 within the IL15 promoter is critical for inhibition of IL15 transcription (Fig. 7A). The methylation analysis conducted using neutrophils from each patient with CTCL as a control also shows that IL15 promoter methylation is relatively specific for the CTCL patient CD4+ T cells, suggesting a model whereby aberrant epigenetic regulation of IL15 expression in CD4+ T cells generates an autocrine loop allowing IL15 to sustain malignant CD4+ cell growth as well as methylation of its IL15 promoter (27). The mechanisms leading to the hypermethylation of the IL15 promoter in human CTCL cells are currently unknown, but could be related to excessive methyltransferase activity (27).

The Il15 tg Mouse as a Model of CTCL

Although the clinical combination of dermatitis, alopecia, pruritus, and skin ulcers is neither specific nor sufficient to define a CTCL phenotype in an animal model, our analysis of the histopathologic and immunophenotypical features of the T-cell lymphoproliferative disorder of Il15 tg mice revealed very compelling parallels with human CTCL. The cutaneous T-cell infiltrate in the Il15 tg mice displayed evident epidermotropism, revealed by the dense infiltrate in the dermal–epidermal junction and by the presence of Pautrier's microabscesses that are pathognomonic for the disease. Furthermore, the T cells isolated from the skin of the mice expressed a set of homing receptors and adhesion molecules that are very typical of the neoplastic, skin-homing T cells of human CTCL. Transplant experiments further support the conclusion that the T-cell neoplasm of the Il15 tg mice predominantly affects the skin, as in human CTCL, because secondary CTCL in SCID mice developed only when T cells, isolated from the skin, but not from the spleen, of affected primary Il15 tg mice were adoptively transplanted. The presence of a mixed CD4+/CD8+ cutaneous T-cell infiltrate, and even dominance of CD8+ T cells in the primary Il15 tg mouse, with oligoclonal rather than monoclonal expansions, also parallels the pattern seen in skin biopsies of patients with early-stage CTCL, where the neoplastic CD4+ T cells are often the minority, CD8+ T cells are overrepresented, and clonality can be difficult to detect (66–68). Interestingly, although the presence of a heavy CD8+ T-cell infiltrate in early, human CTCL has been traditionally interpreted as the sign of a host antitumor response, it is also possible that it may instead reflect polyclonal or oligoclonal, multilineage T-cell expansions in the very early phases of the disease, in response to autocrine production or cross-presentation of IL15 by cognate cells. In advanced-stage CTCL, it is well known that neoplastic CD4+ T cells acquire complex genetic aberrations, expand, outgrow, and possibly suppress CD8+ T cells, developing autocrine growth and metastatic potential. Likewise, secondary CTCL produced by the transplantation of skin lymphocytes from primary Il15 tg mice in our model are more homogeneously CD4+, grow as tumors in the SCID mice, and disseminate, as noted in the late stages of human CTCL. The early development of MF-like disease compared with longer latency for leukemic CTCL in transplanted mice could be attributed to the extent to which accumulation of mutation over time causes the emergence of a more aggressive, clonal leukemic variant.

Regulation of HDACs under Chronic Inflammatory Conditions Such as CTCL

The oncogenic effect of IL15 during the transformation process is mediated by a variety of signaling pathways (15). Chronic exposure to proinflammatory cytokines such as IL15 can have profound impact on the epigenetic signature of the cellular genome (15, 27). Data from patients with CTCL provide clues into the complex epigenome of neoplastic T cells, including altered histone code (69) and aberrant methylation (55). Of histone modifiers, HDACs, especially isoform HDAC1, have been found to be overexpressed in several malignancies and to participate in pathogenesis by repressing tumor-suppressor genes (69, 70). The isoform HDAC1 belongs to the class I family of HDACs and is located in the nucleus. Interaction partners for HDAC1 include HDAC2, p53, RB, MYOD, NF-κB, DNMT1, DNMT3a, MBD2, SP1, BRCA1, MeCP2, ATM, SMAD7, and STAT1 and STAT2 (70, 71). Recently, HDAC1 has become of interest as a potential cancer target because it is recruited by the MDM2 protein and subsequently promotes the degradation of the tumor-suppressor gene TP53 (72, 73). Indeed, in patients with CTCL, HDAC inhibition via pharmacologic inhibitors has yielded very robust therapeutic efficacy (34, 74). Although increased expression of HDAC1 and HDAC6 may have prognostic significance in patients with CTCL (75), factors inducing their upregulation are primarily unknown. In this study, we observed a correlation between overexpression of IL15 and expression of HDAC1 and HDAC6 in CTCL patient CD4+ T cells, and identified IL15 as a factor capable of inducing HDAC expression in normal donor CD4+ T cells in vitro. HDAC overexpression in normal CD4+ T cells likely contributes to their malignant transformation, because HDACs have been shown to act as bona fide oncogenes in many types of human cancer, by regulating the expression of genes involved in cancer initiation and progression through deacetylation of histones (76) as well as nonhistone proteins such as p53 (77). Furthermore, induction of HDACs by IL15 can further amplify tissue inflammation and cell transformation because HDACs positively regulate expression of other proinflammatory cytokines such as IL6, IL8, and TNF (78, 79).

Most noteworthy, while studying regulation of HDACs by IL15, detailed analysis of HDAC1 binding revealed a previously unknown function of HDAC1 as a negative regulator of HDAC6 in normal CD4+ cells. Research from other groups has shown that HDAC1 is associated with a variety of complexes involved in gene silencing, including ones that contain the proteins SIN3 (switch independent protein 3), NuRD (nucleosome remodeling and histone deacetylase), Co-REST (corepressor for REI silencing transcription factor), RB, SP1, and PRC2 (80). HDAC1 is incorporated into the NuRD complex that mediates DNA methylation by recruiting DNMT1 and chromatin remodeling with recruitment of helicase/ATPase family members (80). Although overexpression and improper recruitment of HDACs have been found in cancers (81), the identity of the genomic landscape of HDAC1 in malignancies remains unknown.

Exploring the mechanistic aspects of HDAC regulation by IL15, we observed upregulation of HDAC1 and HDAC6 in normal T cells following exposure to IL15. The involvement of HDAC1 in CTCL is further underscored by our observation that (i) IL15-overexpressing human CTCL CD4+ T cells display decreased binding of HDAC1 at its own promoter, implying loss of the negative autoregulatory loop, which normally controls HDAC1 expression; and (ii) similar changes in HDAC1 binding can be induced by stimulating normal CD4+ T cells with IL15 in vitro. Aberrant IL15 signaling may therefore lead to upregulation of HDAC1 in CTCL cells via loss of the negative autoregulatory loop (35). IL15 also caused similar loss of HDAC1 occupancy at the HDAC6 promoter, resulting in increased HDAC6 in normal CD4+ T cells stimulated with IL15. HDAC6 has been shown to increase angiogenesis and facilitate cell migration, thereby enhancing the metastatic potential (82). In lymphocytes particularly, HDAC6 has been shown to increase cell migration independent of its deacetylase activity (38).

Although HDAC1 is primarily found in repressor complexes, we have uncovered a previously unknown function of HDAC1 as an activator of onco-mir-21, thus playing dual roles in oncogenesis. Detailed analysis of HDAC1 binding revealed increased presence of HDAC1 in miR-21 loci in CTCL patient CD4+ T cells, and in normal donor CD4+ T cells when stimulated with IL15 and compared with resting normal donor CD4+ T cells. With the emerging role of miR-21 in a variety of cancers, it is possible that HDAC1 forms an activating complex to induce miR-21. Interfering with HDAC1 signaling using an HDAC1-specific inhibitor or knockdown of HDAC1 using shRNA supported the importance of HDAC1 in the positive regulation of miR-21. Considering that STAT3 activates miR-21 in patients with CTCL and that IL15 activates both miR-21 and also STAT3 signaling, it will be interesting to further explore the complex interplay between HDAC1 in IL15-mediated oncogenesis (15, 44). Thus, overexpression of IL15 in normal T cells can cause malignant transformation by (1) increasing cell proliferation/survival via repression of tumor-suppressor genes through the induction of core histone deacetylase HDAC1 at their promoter regions, (2) induction of miR-21, a bona fide onco-miR, and (3) possible extravasation of T cells by increasing their migration through HDAC6-mediated deacetylation of tubulin (Fig. 7B). The clinical relevance of these findings is supported by the observation that both pan- and isotype-specific HDACis can prevent progression of CTCL in the Il15 tg mice and in humans (34).

Selective Targeting of HDACs in Il15 tg Mice Impedes Progression of CTCL

Although some of the highest response rates to single-agent pan-HDACis have been observed in patients with CTCL, the overall efficacy of such inhibitors remains limited, in part due to toxicity (34). Both vorinostat and romidepsin are FDA-approved HDACis with favorable clinical activity in the treatment of CTCL; however, response rates are only 30% to 35% (34, 83, 84). In addition to the moderate response rates observed with these drugs, the fact that vorinostat and romidepsin are not HDAC isotype specific but rather pan-HDAC and a class 1 inhibitor, respectively, prevents the identification of the key signaling pathways that must be targeted to improve the efficacy and possibly limit the toxicity of this class of drugs. Il15 tg mice treated with the HDAC6-specific inhibitor WT161 showed delay in progression of CTCL compared with placebo-treated mice, but WT161 was less effective than the HDAC1/2 inhibitor Merck60. Interestingly, the combination of two compounds was less effective than either agent alone in preventing CTCL. As our experimental work largely demonstrates a halt in the progression of early-stage CTCL, we believe this targeted therapeutic might be best applied in a comparable clinical setting.

Human CTCL begins as an indolent neoplasm characterized by the progressive and predominant accumulation of malignant CD4+ T cells in the upper layers of the epidermis, and gradually proceeds to affect larger body surface areas and ultimately to leukemic and visceral spread. Once patients progress to tumor stage, survival is significantly compromised. The key mechanisms initiating and promoting the stepwise progression in CTCL have not been identified, but a set of prior observations and the data presented here support an important role for the dysregulation of IL15. The parallel investigation of CTCL in Il15 tg mice and in CTCL patient samples performed in this study showed a similar pattern where initially small populations of CD4+ T cells are attracted to and retained in the skin microenvironment, and their docking to epidermal Langerhans cells produces the histologic hallmark of CTCL: Pautrier's microabscesses. In patients with CTCL, the oncogenetic aberrations and even the malignant nature of this “founder” population are incompletely understood because of the difficulty to isolate these cells in the midst of a dominant reactive cellular infiltrate. In patients with CTCL, over a variable interval, the continuous presence of microenvironmental factors sustaining lymphocyte trafficking to the skin, combined with the selective drive exerted by repeated exposure to less-than-completely effective therapies, leads to the emergence of clonal populations of T cells with chromosomal instability, complex genetic aberrations, autocrine growth, constitutive activation of NF-κB signaling, and metastatic potential. Finally, we show that a mechanism of IL15 overexpression in patients with CTCL could be related to promoter hypermethylation and the failure of the transcriptional repressor ZEB1 to have access to the IL15 regulatory region. This is a novel finding that not only raises interesting questions about the epigenetic regulation of IL15 in normal individuals and patients with CTCL, but also supports the consideration of utilizing a therapeutic combination of HDACis and demethylating agents in CTCL (85–87).

In summary, we provide in vivo evidence that IL15 likely can have a causal role in the pathogenesis of at least some cases of CTCL, in part via the epigenetic inhibition of the transcriptional repressor ZEB1, which in turn leads to overexpression of IL15 and activation of specific HDACs. Il15 tg CTCL mice provide a novel model for studying the development of CTCL and evaluating potential therapies. Selective inhibition of HDAC1/2 produces a comparable halt in the progression of experimental CTCL to that seen with the pan-HDACis, and thus may provide an equally potent yet less toxic alternative in the clinic.

Human T-cell Isolation

Peripheral blood mononuclear cells were obtained from patients after informed consent in accordance with the Declaration of Helsinki. The Institutional Review Board of Henry Ford Hospital and the Ohio State University Comprehensive Cancer Center approved this study. Normal donor cells were obtained from the Red Cross Blood Bank. The CD4+ T cells from peripheral blood were purified (approximately 95% or more) by negative selection using RosetteSep human CD4 depletion (STEMCELL) as per the manufacturer's instructions.

Mouse Skin T-cell Isolation

All animal studies were performed under approved protocols following the Ohio State University Institutional Animal Care and Use Committee. Mice were housed in a barrier facility. Age- and sex-matched WT, Il15 tg, and SCID mice (The Jackson laboratory) were used. All procedures were done using sterile techniques and tools. Before harvesting skin, all mice were sprayed with 70% isopropanol (Ricca Chemical Company). WT mice were shaved prior to collection of skin sample. Skin was peeled off the mouse at both the dorsal and ventral sides of the skin (approximately 8–12 cm2). The skin was removed and digested in 10 mg/mL of collagenase (Sigma Aldrich) diluted 1:1,000 in RPMI-1640 medium (GIBCO). The tissues were minced using sterile scissors and digested thoroughly for 30 minutes at 37°C (vigorously shaking at 300 rpm for a total of 60 minutes). The supernatant suspension was filtered using a 70-μm nylon mesh cell strainer (BD Bioscience) and then washed with RPMI-1640 medium with 2% FBS at 1,500 rpm for 10 minutes. Lymphocytes were isolated using Ficoll-Paque PLUS (GE Healthcare) gradient centrifugation and then washed with PBS (GIBCO) with 2% FBS. The single-cell suspension was then stained for use in flow cytometry and cell sorting.

Skin Mononuclear Cell Transplantation

After isolation, skin cells were washed twice in 10 mL of PBS and resuspended in 200 μL PBS for transplantation. Injections were administered subcutaneously in the dorsal inferior half of the body using a 1 mL BD Tuberculin syringe with 27 G × ½ BD PrecisionGlide detachable needle after shaving with an electric razor. Mice were observed daily for development of lesions or tumors, and reshaved once every 30 days. Photographs were taken when observable lesions were noteworthy.

Antibody Staining and Flow Cytometry

Peripheral blood, spleen, and skin samples were harvested from moribund mice. Single-cell suspensions were prepared for all samples. The following fluorochrome-conjugated mAbs were purchased from BD Pharmingen and were used for flow cytometry per the manufacturer's instructions: CD8a (clone 53-6.7), CD3e (clone 145-2C11), CD4 (clone RM4-5), CLA/CD162 (clone 2PH1), CD62L (clone MEL-14), CD69 (clone H1.2F3), yδ TCR (clone GL3), Vβ2 (clone B20.6), Vβ3 (clone KJ25), Vβ4 (clone KT4), Vβ5.1 (clone MR9-4), Vβ6 (clone RR4-7), Vβ7 (clone TR310), Vβ8.1 (clone MR5-2), Vβ8.3 (clone 1B3.3), Vβ9 (clone MR10-2), Vβ10b (clone B21.5), Vβ11 (clone RR3-15), Vβ12 (clone MR11-1), Vβ13 (clone MR12-3), Vβ14 (clone 14-2), and Vβ17a (clone KJ23).

Immunofluorescence Staining

Skin sections were frozen in OCT Compound (Sakura Finetek), cryosectioned into 8-μm sections, and placed on glass slides. Skin sections were fixed for 5 minutes in cold acetone, washed twice with PBS, and incubated with protein block (Dako) for 30 minutes at room temperature. Sections were washed twice with PBS and stained for CCR4 or CLA (CD162). Goat polyclonal anti-CCR4 antibody (Abcam, 1664) was used at 1/100 dilution in antibody diluent (Dako) for an hour. Sections were washed 7 times with PBS supplemented with 2% FBS, followed by incubation with 1/500 dilution of donkey anti-Goat IgG H&L (PE) secondary antibody (ab7004) for an hour at room temperature. CLA/CD162 staining was performed similarly by incubating skin sections with 1/00 dilution of mAb CD162 (clone 2PH1) for an hour at room temperature. Skin sections were washed 7 times with PBS and mounted with a drop of VECTASHIELD mounting medium (Vector Laboratories).

Isolation of RNA, cDNA Preparation, and PCR

Purified cells were pelleted and resuspended in TRIzol Reagent (Invitrogen). Total RNA was prepared as previously described (27). RNA was quantified by NanoDrop 1000 Spectrophotometer (Thermo Scientific) and used for cDNA synthesis. cDNA synthesis was achieved by the SuperScript First-Strand Synthesis System for RT-PCR Kit (Invitrogen) using random hexamers as per the manufacturer's protocol. Real-time PCR was performed in a 20-μL volume using 1-μL cDNA, 20X Taqman Assay for each gene, and 2X Taqman Universal Fast PCR Master Mix (Applied Biosystems). An ABI 7900HT Fast Real-Time PCR System (Applied Biosystems) was used for standard fast-protocol for Taqman PCR. Taqman probe ID for the genes can be provided upon request.

Cloning Human IL15 Promoter

Human IL15 was PCR-amplified from CD4+ normal donor genomic DNA for construction of promoter-containing luciferase reporter plasmids. Regions of IL15 gene that contained 500 bp of the promoter region upstream of the transcription start site were amplified and cloned in TOPO TA Cloning Vector. The vector was introduced into TOP 10 One Shot competent bacteria; positive clones were selected and verified by restriction digest with KpnI and XhoI. Released band was purified using the QIAquick Gel Extraction Kit (Qiagen) as per the manufacturer's instructions. The gel purified DNA fragments containing IL15 promoter were reinserted into pGL3-basic luciferase vector (Promega) and verified by restriction enzyme digestion.

Transfection and Luciferase Assay

Approximately 5 × 106 Jurkat cells (obtained from the ATCC) were transfected with 2.5 μg of methylated or nonmethylated plasmid constructs and 0.5 μg of renilla luciferase plasmid using the amaxa human T-cell transfection kit (Lonza). Transfection was performed using the manufacturer's protocol using the V-24 program. Cells were harvested and lysed 16 to 24 hours after transfection for luciferase assay using the Dual-Luciferase Reporter Assay System (Promega).

In Vitro Culture of CD4+ T Cells with IL15

Purified CD4+ T cells from normal donors were seeded in a 24-well plate at a density of 1 × 106 cells per well. Cells were incubated at 37°C and 5% CO2 in RPMI-1640 and chronically stimulated with IL15 at a concentration of 100 ng/mL. Cells were harvested at indicated time point of incubation for either immunoblot analysis or ChIP assay. For growth analysis of T cells in response to IL15, long-term culture was maintained by replacing 0.5 mL of old medium with fresh medium containing IL15.

Transfection and Migration Assay

Approximately 10 × 106 freshly purified CD4+ cells were transfected with 10 μg of shRNA plasmid as described above. Forty-eight hours after transfection, cells were FACS sorted for GFP expression. GFP+ cells were seeded in the upper half of a cell-permeable membrane cassette, and cells were allowed to migrate toward the lower chamber containing serum-free medium supplemented with 50 ng/mL of IL15 for 12 hours. Cells in the lower chamber were counted by trypan blue exclusion, and relative percentage migration was calculated by subtracting spontaneous migration.

In Vitro Drug Treatment and Cell Viability Assay

Hut78 cells (obtained from the ATCC) were seeded at a density of 1 × 105 cells/well in a 96-well plate in triplicate. Drugs were added at the indicated time point to cells on day 0. Cell viability was determined by MTS assay using CellTiter 96 Aqueous One Solution Reagent (Promega) at 48 to 72 hours as per the manufacturer's instructions.

HDACi Therapy in CTCL Mice

In order to evaluate the effects of inhibiting HDAC1/2 and HDAC6 in progression of CTCL, 4-week-old IL15 tg mice were treated with 50 mg/kg of JQ12 and/or WT161, respectively. Control mice were treated with equal volume of 10% DMSO in saline. Mice were given intraperitoneal injection 5 days/week for 4 weeks. Mice were sacrificed and analyzed 2 days after cessation of treatment. The mice were observed daily for changes in severity of lesions, and photographs were taken at the beginning and end of the therapy. Mice were euthanized and analyzed for malignant infiltration in skin by histology analysis.

Epigenetic Analysis

DNA methylation on patient and normal donor CD4+ cells was performed by EpigenDx Inc. ChIP-sequencing studies were performed using FactorPath ChIP-Seq Services (Active Motif).

Statistical Analysis

Two-sample t test was used to compare two independent groups, and paired t test was used to compare two paired groups. Data transformation was performed if the original distribution is non-normal. ANOVA models or generalized linear models were used to compare three or more groups. Survival curves were estimated by the Kaplan–Meier method, and treatment groups were compared by the log-rank test. P values were adjusted for multiple comparisons by Holm's procedure. A P value of <0.05 was considered significant.

H. Wong is a consultant/advisory board member for Actelion and Seattle Genetics. J.E. Bradner is President at Novartis Institute of BioMedical Research. P. Porcu reports receiving a commercial research grant from Galderma. No potential conflicts of interest were disclosed by the other authors.

Conception and design: A. Mishra, G. Marcucci, P. Porcu, M.A. Caligiuri

Development of methodology: A. Mishra, J. Wen, G. Marcucci, J.E. Bradner, P. Porcu

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A. Mishra, S. Kwiatkowski, L.A. Sullivan, G.H. Sams, J. Johns, D.P. Curphey, K. McConnell, H. Wong, G. Russo, P. Porcu

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): A. Mishra, K. La Perle, S. Kwiatkowski, L.A. Sullivan, G.H. Sams, D.P. Curphey, J. Zhang, G. Marcucci, J.E. Bradner, P. Porcu, M.A. Caligiuri

Writing, review, and/or revision of the manuscript: A. Mishra, L.A. Sullivan, G.H. Sams, D.P. Curphey, G. Marcucci, P. Porcu, M.A. Caligiuri

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): A. Mishra, S. Kwiatkowski, L.A. Sullivan, G.H. Sams, J. Johns, D.P. Curphey, J. Qi

Study supervision: A. Mishra, P. Porcu, M.A. Caligiuri

Other (comparative pathologic evaluation of mice; histopathologic interpretation of skin and organs, including routine and immunohistochemical stains; preparation of photographs): K. La Perle

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Willemze
R
,
Jaffe
ES
,
Burg
G
,
Cerroni
L
,
Berti
E
,
Swerdlow
SH
, et al
WHO-EORTC classification for cutaneous lymphomas
.
Blood
2005
;
105
:
3768
85
.
2.
Olsen
E
,
Vonderheid
E
,
Pimpinelli
N
,
Willemze
R
,
Kim
Y
,
Knobler
R
, et al
Revisions to the staging and classification of mycosis fungoides and Sezary syndrome: A proposal of the International Society for Cutaneous Lymphomas (ISCL) and the cutaneous lymphoma task force of the European Organization of Research and Treatment of Cancer (EORTC)
.
Blood
2007
;
110
:
1713
22
.
3.
Duvic
M
,
Talpur
R
,
Ni
X
,
Zhang
C
,
Hazarika
P
,
Kelly
C
, et al
Phase 2 trial of oral vorinostat (suberoylanilide hydroxamic acid, SAHA) for refractory cutaneous T-cell lymphoma (CTCL)
.
Blood
2007
;
109
:
31
9
.
4.
Whittaker
SJ
,
Demierre
MF
,
Kim
EJ
,
Rook
AH
,
Lerner
A
,
Duvic
M
, et al
Final results from a multicenter, international, pivotal study of romidepsin in refractory cutaneous T-cell lymphoma
.
J Clin Oncol
2010
;
28
:
4485
91
.
5.
Yamanaka
K
,
Clark
R
,
Rich
B
,
Dowgiert
R
,
Hirahara
K
,
Hurwitz
D
, et al
Skin-derived interleukin-7 contributes to the proliferation of lymphocytes in cutaneous T-cell lymphoma
.
Blood
2006
;
107
:
2440
5
.
6.
Singer
EM
,
Shin
DB
,
Nattkemper
LA
,
Benoit
BM
,
Klein
RS
,
Didigu
CA
, et al
IL-31 is produced by the malignant T-cell population in cutaneous T-Cell lymphoma and correlates with CTCL pruritus
.
J Invest Dermatol
2013
;
133
:
2783
5
.
7.
Krejsgaard
T
,
Litvinov
IV
,
Wang
Y
,
Xia
L
,
Willerslev-Olsen
A
,
Koralov
SB
, et al
Elucidating the role of interleukin-17F in cutaneous T-cell lymphoma
.
Blood
2013
;
122
:
943
50
.
8.
Guenova
E
,
Watanabe
R
,
Teague
JE
,
Desimone
JA
,
Jiang
Y
,
Dowlatshahi
M
, et al
TH2 cytokines from malignant cells suppress TH1 responses and enforce a global TH2 bias in leukemic cutaneous T-cell lymphoma
.
Clin Cancer Res
2013
;
19
:
3755
63
.
9.
Geskin
LJ
,
Viragova
S
,
Stolz
DB
,
Fuschiotti
P
. 
Interleukin-13 is overexpressed in cutaneous T-cell lymphoma cells and regulates their proliferation
.
Blood
2015
;
125
:
2798
805
.
10.
Asadullah
K
,
Haeussler-Quade
A
,
Gellrich
S
,
Hanneken
S
,
Hansen-Hagge
TE
,
Docke
WD
, et al
IL-15 and IL-16 overexpression in cutaneous T-cell lymphomas: Stage-dependent increase in mycosis fungoides progression
.
Exp Dermatol
2000
;
9
:
248
51
.
11.
Wasik
MA
. 
IL-13 as a novel growth factor in CTCL
.
Blood
2015
;
125
:
2737
8
.
12.
Vowels
BR
,
Lessin
SR
,
Cassin
M
,
Jaworsky
C
,
Benoit
B
,
Wolfe
JT
, et al
Th2 cytokine mRNA expression in skin in cutaneous T-cell lymphoma
.
J Invest Dermatol
1994
;
103
:
669
73
.
13.
Krejsgaard
T
,
Ralfkiaer
U
,
Clasen-Linde
E
,
Eriksen
KW
,
Kopp
KL
,
Bonefeld
CM
, et al
Malignant cutaneous T-cell lymphoma cells express IL-17 utilizing the Jak3/Stat3 signaling pathway
.
J Invest Dermatol
2011
;
131
:
1331
8
.
14.
Dobbeling
U
,
Dummer
R
,
Laine
E
,
Potoczna
N
,
Qin
JZ
,
Burg
G
. 
Interleukin-15 is an autocrine/paracrine viability factor for cutaneous T-cell lymphoma cells
.
Blood
1998
;
92
:
252
8
.
15.
Mishra
A
,
Sullivan
L
,
Caligiuri
MA
. 
Molecular pathways: Interleukin-15 signaling in health and in cancer
.
Clin Cancer Res
2014
;
20
:
2044
50
.
16.
Fehniger
TA
,
Caligiuri
MA
. 
Interleukin 15: Biology and relevance to human disease
.
Blood
2001
;
97
:
14
32
.
17.
Leroy
S
,
Dubois
S
,
Tenaud
I
,
Chebassier
N
,
Godard
A
,
Jacques
Y
, et al
Interleukin-15 expression in cutaneous T-cell lymphoma (mycosis fungoides and Sezary syndrome)
.
Br J Dermatol
2001
;
144
:
1016
23
.
18.
Marzec
M
,
Halasa
K
,
Kasprzycka
M
,
Wysocka
M
,
Liu
X
,
Tobias
JW
, et al
Differential effects of interleukin-2 and interleukin-15 versus interleukin-21 on CD4+ cutaneous T-cell lymphoma cells
.
Cancer Res
2008
;
68
:
1083
91
.
19.
Qin
JZ
,
Zhang
CL
,
Kamarashev
J
,
Dummer
R
,
Burg
G
,
Dobbeling
U
. 
Interleukin-7 and interleukin-15 regulate the expression of the bcl-2 and c-myb genes in cutaneous T-cell lymphoma cells
.
Blood
2001
;
98
:
2778
83
.
20.
Han
GW
,
Iwatsuki
K
,
Inoue
M
,
Matsui
T
,
Nishibu
A
,
Akiba
H
, et al
Interleukin-15 is not a constitutive cytokine in the epidermis, but is inducible in culture or inflammatory conditions
.
Acta Derm Venereol
1999
;
79
:
37
40
.
21.
Marzec
M
,
Liu
X
,
Kasprzycka
M
,
Witkiewicz
A
,
Raghunath
PN
,
El-Salem
M
, et al
IL-2- and IL-15-induced activation of the rapamycin-sensitive mTORC1 pathway in malignant CD4+ T lymphocytes
.
Blood
2008
;
111
:
2181
9
.
22.
Kasprzycka
M
,
Zhang
Q
,
Witkiewicz
A
,
Marzec
M
,
Potoczek
M
,
Liu
X
, et al
Gamma c-signaling cytokines induce a regulatory T cell phenotype in malignant CD4+ T lymphocytes
.
J Immunol
2008
;
181
:
2506
12
.
23.
Adachi
T
,
Kobayashi
T
,
Sugihara
E
,
Yamada
T
,
Ikuta
K
,
Pittaluga
S
, et al
Hair follicle-derived IL-7 and IL-15 mediate skin-resident memory T cell homeostasis and lymphoma
.
Nat Med
2015
;
21
:
1272
9
.
24.
Wang
J
,
Lee
S
,
Teh
CE
,
Bunting
K
,
Ma
L
,
Shannon
MF
. 
The transcription repressor, ZEB1, cooperates with CtBP2 and HDAC1 to suppress IL-2 gene activation in T cells
.
Int Immunol
2009
;
21
:
227
35
.
25.
Sanchez-Tillo
E
,
de Barrios
O
,
Siles
L
,
Amendola
PG
,
Darling
DS
,
Cuatrecasas
M
, et al
ZEB1 Promotes invasiveness of colorectal carcinoma cells through the opposing regulation of uPA and PAI-1
.
Clin Cancer Res
2013
;
19
:
1071
82
.
26.
Fehniger
TA
,
Suzuki
K
,
Ponnappan
A
,
VanDeusen
JB
,
Cooper
MA
,
Florea
SM
, et al
Fatal leukemia in interleukin 15 transgenic mice follows early expansions in natural killer and memory phenotype CD8+ T cells
.
J Exp Med
2001
;
193
:
219
31
.
27.
Mishra
A
,
Liu
S
,
Sams
GH
,
Curphey
DP
,
Santhanam
R
,
Rush
LJ
, et al
Aberrant overexpression of IL-15 initiates large granular lymphocyte leukemia through chromosomal instability and DNA hypermethylation
.
Cancer Cell
2012
;
22
:
645
55
.
28.
Edelson
RL
. 
Cutaneous T cell lymphoma: The helping hand of dendritic cells
.
Ann N Y Acad Sci
2001
;
941
:
1
11
.
29.
Cornejo
MG
,
Kharas
MG
,
Werneck
MB
,
Le Bras
S
,
Moore
SA
,
Ball
B
, et al
Constitutive JAK3 activation induces lymphoproliferative syndromes in murine bone marrow transplantation models
.
Blood
2009
;
113
:
2746
54
.
30.
Whittaker
S
. 
Biological insights into the pathogenesis of cutaneous T-cell lymphomas (CTCL)
.
Semin Oncol
2006
;
33
(
1 Suppl 3
):
S3
6
.
31.
Borowitz
MJ
,
Weidner
A
,
Olsen
EA
,
Picker
LJ
. 
Abnormalities of circulating T-cell subpopulations in patients with cutaneous T-cell lymphoma: cutaneous lymphocyte-associated antigen expression on T cells correlates with extent of disease
.
Leukemia
1993
;
7
:
859
63
.
32.
O'Connor
OA
,
Toner
LE
,
Vrhovac
R
,
Budak-Alpdogan
T
,
Smith
EA
,
Bergman
P
. 
Comparative animal models for the study of lymphohematopoietic tumors: Strengths and limitations of present approaches
.
Leuk Lymphoma
2005
;
46
:
973
92
.
33.
Weinberg
JM
,
Jaworsky
C
,
Benoit
BM
,
Telegan
B
,
Rook
AH
,
Lessin
SR
. 
The clonal nature of circulating Sezary cells
.
Blood
1995
;
86
:
4257
62
.
34.
Rangwala
S
,
Zhang
C
,
Duvic
M
. 
HDAC inhibitors for the treatment of cutaneous T-cell lymphomas
.
Future Med Chem
2012
;
4
:
471
86
.
35.
Schuettengruber
B
,
Simboeck
E
,
Khier
H
,
Seiser
C
. 
Autoregulation of mouse histone deacetylase 1 expression
.
Mol Cell Biol
2003
;
23
:
6993
7004
.
36.
Johnstone
RW
,
Licht
JD
. 
Histone deacetylase inhibitors in cancer therapy: Is transcription the primary target?
Cancer Cell
2003
;
4
:
13
8
.
37.
Wilting
RH
,
Yanover
E
,
Heideman
MR
,
Jacobs
H
,
Horner
J
,
van der Torre
J
, et al
Overlapping functions of Hdac1 and Hdac2 in cell cycle regulation and haematopoiesis
.
EMBO J
2010
;
29
:
2586
97
.
38.
Cabrero
JR
,
Serrador
JM
,
Barreiro
O
,
Mittelbrunn
M
,
Naranjo-Suarez
S
,
Martin-Cofreces
N
, et al
Lymphocyte chemotaxis is regulated by histone deacetylase 6, independently of its deacetylase activity
.
Mol Biol Cell
2006
;
17
:
3435
45
.
39.
Creyghton
MP
,
Cheng
AW
,
Welstead
GG
,
Kooistra
T
,
Carey
BW
,
Steine
EJ
, et al
Histone H3K27ac separates active from poised enhancers and predicts developmental state
.
Proc Natl Acad Sci U S A
2010
;
107
:
21931
6
.
40.
Hsu
PY
,
Hsu
HK
,
Lan
X
,
Juan
L
,
Yan
PS
,
Labanowska
J
, et al
Amplification of distant estrogen response elements deregulates target genes associated with tamoxifen resistance in breast cancer
.
Cancer Cell
2013
;
24
:
197
212
.
41.
Medina
PP
,
Nolde
M
,
Slack
FJ
. 
OncomiR addiction in an in vivo model of microRNA-21-induced pre-B-cell lymphoma
.
Nature
2010
;
467
:
86
90
.
42.
Narducci
MG
,
Arcelli
D
,
Picchio
MC
,
Lazzeri
C
,
Pagani
E
,
Sampogna
F
, et al
MicroRNA profiling reveals that miR-21, miR486 and miR-214 are upregulated and involved in cell survival in Sezary syndrome
.
Cell Death Dis
2011
;
2
:
e151
.
43.
Sandoval
J
,
Diaz-Lagares
A
,
Salgado
R
,
Servitje
O
,
Climent
F
,
Ortiz-Romero
PL
, et al
MicroRNA expression profiling and DNA methylation signature for deregulated microRNA in cutaneous T-cell lymphoma
.
J Invest Dermatol
2015
;
135
:
1128
37
.
44.
van der Fits
L
,
van Kester
MS
,
Qin
Y
,
Out-Luiting
JJ
,
Smit
F
,
Zoutman
WH
, et al
MicroRNA-21 expression in CD4+ T cells is regulated by STAT3 and is pathologically involved in Sezary syndrome
.
J Invest Dermatol
2011
;
131
:
762
8
.
45.
Batty
N
,
Malouf
GG
,
Issa
JP
. 
Histone deacetylase inhibitors as anti-neoplastic agents
.
Cancer Lett
2009
;
280
:
192
200
.
46.
Methot
JL
,
Chakravarty
PK
,
Chenard
M
,
Close
J
,
Cruz
JC
,
Dahlberg
WK
, et al
Exploration of the internal cavity of histone deacetylase (HDAC) with selective HDAC1/HDAC2 inhibitors (SHI-1:2)
.
Bioorg Med Chem Lett
2008
;
18
:
973
8
.
47.
Guan
JS
,
Haggarty
SJ
,
Giacometti
E
,
Dannenberg
JH
,
Joseph
N
,
Gao
J
, et al
HDAC2 negatively regulates memory formation and synaptic plasticity
.
Nature
2009
;
459
:
55
60
.
48.
Malamut
G
,
El Machhour
R
,
Montcuquet
N
,
Martin-Lanneree
S
,
Dusanter-Fourt
I
,
Verkarre
V
, et al
IL-15 triggers an antiapoptotic pathway in human intraepithelial lymphocytes that is a potential new target in celiac disease-associated inflammation and lymphomagenesis
.
J Clin Invest
2010
;
120
:
2131
43
.
49.
Wu
S
,
Fischer
L
,
Gokbuget
N
,
Schwartz
S
,
Burmeister
T
,
Notter
M
, et al
Expression of interleukin 15 in primary adult acute lymphoblastic leukemia
.
Cancer
2010
;
116
:
387
92
.
50.
Gazdar
AF
,
Carney
DN
,
Bunn
PA
,
Russell
EK
,
Jaffe
ES
,
Schechter
GP
, et al
Mitogen requirements for the in vitro propagation of cutaneous T-cell lymphomas
.
Blood
1980
;
55
:
409
17
.
51.
Poiesz
BJ
,
Ruscetti
FW
,
Gazdar
AF
,
Bunn
PA
,
Minna
JD
,
Gallo
RC
. 
Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma
.
Proc Natl Acad Sci U S A
1980
;
77
:
7415
9
.
52.
Bamford
RN
,
Battiata
AP
,
Burton
JD
,
Sharma
H
,
Waldmann
TA
. 
Interleukin (IL) 15/IL-T production by the adult T-cell leukemia cell line HuT-102 is associated with a human T-cell lymphotrophic virus type I region/IL-15 fusion message that lacks many upstream AUGs that normally attenuates IL-15 mRNA translation
.
Proc Natl Acad Sci U S A
1996
;
93
:
2897
902
.
53.
Das
PM
,
Singal
R
. 
DNA methylation and cancer
.
J Clin Oncol
2004
;
22
:
4632
42
.
54.
Lewis
J
,
Bird
A
. 
DNA methylation and chromatin structure
.
FEBS Lett
1991
;
285
:
155
9
.
55.
van Doorn
R
,
Zoutman
WH
,
Dijkman
R
,
de Menezes
RX
,
Commandeur
S
,
Mulder
AA
, et al
Epigenetic profiling of cutaneous T-cell lymphoma: Promoter hypermethylation of multiple tumor suppressor genes including BCL7a, PTPRG, and p73
.
J Clin Oncol
2005
;
23
:
3886
96
.
56.
Contassot
E
,
French
LE
. 
Epigenetic causes of apoptosis resistance in cutaneous T-cell lymphomas
.
J Invest Dermatol
2010
;
130
:
922
4
.
57.
Bahar Halpern
K
,
Vana
T
,
Walker
MD
. 
Paradoxical role of DNA methylation in activation of FoxA2 gene expression during endoderm development
.
J Biol Chem
2014
;
289
:
23882
92
.
58.
Pipaon
C
,
Real
PJ
,
Fernandez-Luna
JL
. 
Defective binding of transcriptional repressor ZEB via DNA methylation contributes to increased constitutive levels of p73 in Fanconi anemia cells
.
FEBS Lett
2005
;
579
:
4610
4
.
59.
de Wilde
J
,
Kooter
JM
,
Overmeer
RM
,
Claassen-Kramer
D
,
Meijer
CJ
,
Snijders
PJ
, et al
hTERT promoter activity and CpG methylation in HPV-induced carcinogenesis
.
BMC Cancer
2010
;
10
:
271
.
60.
Rojas-Marquez
C
,
Valle-Rios
R
,
Lopez-Bayghen
E
,
Ortiz-Navarrete
V
. 
CRTAM is negatively regulated by ZEB1 in T cells
.
Mol Immunol
2015
;
66
:
290
8
.
61.
Choi
J
,
Goh
G
,
Walradt
T
,
Hong
BS
,
Bunick
CG
,
Chen
K
, et al
Genomic landscape of cutaneous T cell lymphoma
.
Nat Genet
2015
;
47
:
1011
9
.
62.
Vermeer
MH
,
van Doorn
R
,
Dijkman
R
,
Mao
X
,
Whittaker
S
,
van Voorst Vader
PC
, et al
Novel and highly recurrent chromosomal alterations in Sezary syndrome
.
Cancer Res
2008
;
68
:
2689
98
.
63.
Williams
TM
,
Moolten
D
,
Burlein
J
,
Romano
J
,
Bhaerman
R
,
Godillot
A
, et al
Identification of a zinc finger protein that inhibits IL-2 gene expression
.
Science
1991
;
254
:
1791
4
.
64.
Hidaka
T
,
Nakahata
S
,
Hatakeyama
K
,
Hamasaki
M
,
Yamashita
K
,
Kohno
T
, et al
Down-regulation of TCF8 is involved in the leukemogenesis of adult T-cell leukemia/lymphoma
.
Blood
2008
;
112
:
383
93
.
65.
Fedele
M
,
Pentimalli
F
,
Baldassarre
G
,
Battista
S
,
Klein-Szanto
AJ
,
Kenyon
L
, et al
Transgenic mice overexpressing the wild-type form of the HMGA1 gene develop mixed growth hormone/prolactin cell pituitary adenomas and natural killer cell lymphomas
.
Oncogene
2005
;
24
:
3427
35
.
66.
Ponti
R
,
Quaglino
P
,
Novelli
M
,
Fierro
MT
,
Comessatti
A
,
Peroni
A
, et al
T-cell receptor gamma gene rearrangement by multiplex polymerase chain reaction/heteroduplex analysis in patients with cutaneous T-cell lymphoma (mycosis fungoides/Sezary syndrome) and benign inflammatory disease: correlation with clinical, histological and immunophenotypical findings
.
Br J Dermatol
2005
;
153
:
565
73
.
67.
Hoppe
RT
,
Medeiros
LJ
,
Warnke
RA
,
Wood
GS
. 
CD8-positive tumor-infiltrating lymphocytes influence the long-term survival of patients with mycosis fungoides
.
J Am Acad Dermatol
1995
;
32
:
448
53
.
68.
Vermeer
MH
,
van Doorn
R
,
Dukers
D
,
Bekkenk
MW
,
Meijer
CJ
,
Willemze
R
. 
CD8+ T cells in cutaneous T-cell lymphoma: Expression of cytotoxic proteins, Fas Ligand, and killing inhibitory receptors and their relationship with clinical behavior
.
J Clin Oncol
2001
;
19
:
4322
9
.
69.
Glozak
MA
,
Seto
E
. 
Histone deacetylases and cancer
.
Oncogene
2007
;
26
:
5420
32
.
70.
Kelly Richard
DW
,
Cowley Shaun
M
. 
The physiological roles of histone deacetylase (HDAC) 1 and 2: Complex co-stars with multiple leading parts
.
Biochem Soc Trans
2013
;
41
:
741
9
.
71.
West
AC
,
Johnstone
RW
. 
New and emerging HDAC inhibitors for cancer treatment
.
J Clin Invest
2014
;
124
:
30
9
.
72.
Arora
A
,
Gera
S
,
Maheshwari
T
,
Raghav
D
,
Alam
MJ
,
Singh
RK
, et al
The dynamics of stress p53-Mdm2 network regulated by p300 and HDAC1
.
PLoS One
2013
;
8
:
e52736
.
73.
Ito
A
,
Kawaguchi
Y
,
Lai
CH
,
Kovacs
JJ
,
Higashimoto
Y
,
Appella
E
, et al
MDM2-HDAC1-mediated deacetylation of p53 is required for its degradation
.
EMBO J
2002
;
21
:
6236
45
.
74.
Zhang
C
,
Richon
V
,
Ni
X
,
Talpur
R
,
Duvic
M
. 
Selective induction of apoptosis by histone deacetylase inhibitor SAHA in cutaneous T-cell lymphoma cells: Relevance to mechanism of therapeutic action
.
J Invest Dermatol
2005
;
125
:
1045
52
.
75.
Marquard
L
,
Gjerdrum
LM
,
Christensen
IJ
,
Jensen
PB
,
Sehested
M
,
Ralfkiaer
E
. 
Prognostic significance of the therapeutic targets histone deacetylase 1, 2, 6 and acetylated histone H4 in cutaneous T-cell lymphoma
.
Histopathology
2008
;
53
:
267
77
.
76.
Jenuwein
T
,
Allis
CD
. 
Translating the histone code
.
Science
2001
;
293
:
1074
80
.
77.
Gu
W
,
Roeder
RG
. 
Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain
.
Cell
1997
;
90
:
595
606
.
78.
Leoni
F
,
Zaliani
A
,
Bertolini
G
,
Porro
G
,
Pagani
P
,
Pozzi
P
, et al
The antitumor histone deacetylase inhibitor suberoylanilide hydroxamic acid exhibits antiinflammatory properties via suppression of cytokines
.
Proc Natl Acad Sci U S A
2002
;
99
:
2995
3000
.
79.
Villagra
A
,
Sotomayor
EM
,
Seto
E
. 
Histone deacetylases and the immunological network: implications in cancer and inflammation
.
Oncogene
2010
;
29
:
157
73
.
80.
Laugesen
A
,
Helin
K
. 
Chromatin repressive complexes in stem cells, development, and cancer
.
Cell Stem Cell
2014
;
14
:
735
51
.
81.
Kelly
RD
,
Cowley
SM
. 
The physiological roles of histone deacetylase (HDAC) 1 and 2: Complex co-stars with multiple leading parts
.
Biochem Soc Trans
2013
;
41
:
741
9
.
82.
Witt
O
,
Deubzer
HE
,
Milde
T
,
Oehme
I
. 
HDAC family: What are the cancer relevant targets?
Cancer Lett
2009
;
277
:
8
21
.
83.
Olsen
EA
,
Kim
YH
,
Kuzel
TM
,
Pacheco
TR
,
Foss
FM
,
Parker
S
, et al
Phase IIb multicenter trial of vorinostat in patients with persistent, progressive, or treatment refractory cutaneous T-cell lymphoma
.
J Clin Oncol
2007
;
25
:
3109
15
.
84.
Piekarz
RL
,
Frye
R
,
Turner
M
,
Wright
JJ
,
Allen
SL
,
Kirschbaum
MH
, et al
Phase II multi-institutional trial of the histone deacetylase inhibitor romidepsin as monotherapy for patients with cutaneous T-cell lymphoma
.
J Clin Oncol
2009
;
27
:
5410
7
.
85.
Singh
BN
,
Zhou
H
,
Li
J
,
Tipton
T
,
Wang
B
,
Shao
G
, et al
Preclinical studies on histone deacetylase inhibitors as therapeutic reagents for endometrial and ovarian cancers
.
Future Oncol
2011
;
7
:
1415
28
.
86.
Suzuki
H
,
Gabrielson
E
,
Chen
W
,
Anbazhagan
R
,
van Engeland
M
,
Weijenberg
MP
, et al
A genomic screen for genes upregulated by demethylation and histone deacetylase inhibition in human colorectal cancer
.
Nat Genet
2002
;
31
:
141
9
.
87.
Thurn
KT
,
Thomas
S
,
Moore
A
,
Munster
PN
. 
Rational therapeutic combinations with histone deacetylase inhibitors for the treatment of cancer
.
Future Oncol
2011
;
7
:
263
83
.

Supplementary data