Abstract
Cutaneous T-cell lymphoma (CTCL) is a malignancy of mature CD4+ T-cells that proliferate in the skin. With advanced disease some patients may experience progression to an aggressive form in which neoplastic cells disseminate to the blood. A hallmark feature in the development and progression of CTCL is the global dysregulation of epigenetic modifications resulting in aberrant gene expression, increased expression of oncogenes, and silencing of tumor suppressors. Therapeutic strategies which target the epigenome have been effective in patients with advanced-stage disease, however short duration of response and development of resistance remain important clinical problems. In this study, we sought to probe the aberrant epigenetic modifications in malignant T-cells and identify potential novel targets for improved therapies.
Oncogenic microRNA (miR)-214 has been shown to be significantly upregulated in patients with advanced CTCL, and this overexpression is associated with worse outcomes. The mechanisms by which miR-214 is upregulated in CTCL are unknown. Utilizing CTCL patient-derived cell lines, primary patient samples, and the recently characterized interleukin-15 (IL-15) transgenic mouse model of CTCL, we describe the regulation of miR-214 expression by bromodomain-containing protein 4 (BRD4) as well as aberrantly-expressed TWIST1. We also describe the efficacy of a specific inhibitor of miR-214 in vivo.
We recently described increased global binding occupancy by BRD4 in CTCL patients, resulting in oncogene overexpression (Kohnken, R. et al. Blood 128:1097, 2016). Here, we show that BRD4 binding occupancy in CTCL patient cells is increased at the promoter region of miR-214. We confirm significant overexpression of miR-214 in CD4+ T-cells in CTCL patients) compared to healthy donors (Figure 1A, mean ± SEM of expression in CTCL patients vs healthy donor CD4+ cells= 10.18 ± 1.22, n=7 vs 1.03 ± 0.06, n=6, unpaired t-test, p=0.0028). Specific inhibition of BRD4 by small molecule JQ1 significantly decreased miR-214 in both patient CD4+ T-cells (0.58 ± 0.008 compared to 1.00 ± 0.003 in control-treated cells, p<0.0001), and in CTCL patient-derived cell lines (0.19 ± 0.01 compared to 1.0 ±0.02 at 1µM JQ1, p<0.0001).
BRD4 has been shown to bind to di-acetylated TWIST1 in several cell types. TWIST1, a cellular regulatory protein often associated with epithelial-to-mesenchymal transition, is not normally expressed in CD4+ T-lymphocytes. Previous work from our lab has demonstrated aberrant overexpression of TWIST1 in CTCL patient CD4+ T-cells secondary to promoter hypomethylation. However, the role of TWIST1 expression in CTCL development has not been explored. We demonstrate that specific silencing of TWIST1 alone (0.51 ± 0.06, p=0.0046 vs 1.0 ± 0.054 for scrambled control), as well as in combination with silencing of BRD4 (0.51 ± 0.005, p=0.0013 vs scrambled control), results in significant decrease in miR-214 levels. Furthermore, we identify a novel target of miR-214, Kruppel-like factor 12 (KLF12), a protein with tumor suppressive functions in other cell types, but whose role in lymphoma is not known.
Next, we performed in vivo treatment of IL-15 transgenic CTCL mice with 25mg/kg antagomiR-214 (n=4) or scrambled control (n=4) beginning at 8 weeks of age for 2 weeks. Scoring of the morphology and severity of skin lesions grossly and histologically demonstrated significant reduction in the extent of neoplastic disease in the skin of mice treated with antagomiR-214 (Figure 1B, C, mean ± SEM of expression in antagomir-214-treated mice vs vehicle-treated mice= 3.75 ± 0.239 vs 6.5 ± 0.144 out of a possible total score of 7, unpaired t-test, p=0.0027).
We conclude that miR-214 overexpression in CTCL is in part driven by increased binding of TWIST1 and subsequent recruitment of BRD4 to the miR-214 promoter region. Specific targeting of miR-214 in vivo using an antagomiR results in reduced disease severity in a transgenic mouse model of CTCL. MiR-214 therefore represents a novel approach and a rational therapeutic target in CTCL.
Porcu: Tetralogic: Research Funding; Kiowa: Research Funding; Innate Pharma: Research Funding; Miragen: Research Funding; Galderma: Research Funding; Kura: Research Funding; Cell Medica: Research Funding; Celgene: Research Funding.
