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Mechanism, Consequences, and Therapeutic Targeting of Abnormal IL15 Signaling in Cutaneous T-cell Lymphoma.

ABSTRACT: 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.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.

SUBMITTER: Mishra A

PROVIDER: S-EPMC5388135 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Mechanism, Consequences, and Therapeutic Targeting of Abnormal IL15 Signaling in Cutaneous T-cell Lymphoma.

Mishra Anjali A La Perle Krista K Kwiatkowski Sonya S Sullivan Laura A LA Sams Gregory H GH Johns Jessica J Curphey Douglas P DP Wen Jing J McConnell Kathleen K Qi Jun J Wong Henry H Russo Giandomenico G Zhang Jianying J Marcucci Guido G Bradner James E JE Porcu Pierluigi P Caligiuri Michael A MA

Cancer discovery 20160715 9

<h4>Unlabelled</h4>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 I ...[more]

PMID: 27422033

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Project description:BackgroundTargeting epigenetic modifiers is effective in cutaneous T cell lymphoma (CTCL). However, there is a need for further improvement of this therapeutic approach. Here, we compared the mode of action of romidepsin (FK228), an established class I histone deacetylase inhibitor, and domatinostat (4SC-202), a novel inhibitor of class I HDACs, which has been reported to also target the lysine-specific histone demethylase 1A (LSD1).MethodsWe performed MTS assays and flow cytometric analyses of propidium iodide or annexin V-stained cells to assess drug impact on cellular proliferation, cell cycle distribution, and survival. Histone acetylation and methylation as well as caspase activation was analyzed by immunoblot. Gene expression analysis was performed using NanosString technology. Knockdown and knockout of LSD1 was achieved with shRNA and CRISPR/Cas9, respectively, while the CRISPR/Cas9 synergistic activation mediator system was used to induce expression of endogenous HDACs and LSD1. Furthermore, time-lapse fluorescence microscopy and an in vitro tubulin polymerization assay were applied.ResultsWhile FK228 as well as 4SC-202 potently induced cell death in six different CTCL cell lines, only in the case of 4SC-202 death was preceded by an accumulation of cells in the G2/M phase of the cell cycle. Surprisingly, apoptosis and accumulation of cells with double DNA content occurred already at 4SC-202 concentrations hardly affecting histone acetylation and methylation, and provoking significantly less changes in gene expression compared to biologically equivalent doses of FK228. Indeed, we provide evidence that the 4SC-202-induced G2/M arrest in CTCL cells is independent of de novo transcription. Furthermore, neither enforced expression of HDAC1 nor knockdown or knockout of LSD1 affected the 4SC-202-induced effects. Since time-lapse microscopy revealed that 4SC-202 could affect mitotic spindle formation, we performed an in vitro tubulin polymerization assay revealing that 4SC-202 can directly inhibit microtubule formation.ConclusionsWe demonstrate that 4SC-202, a drug currently tested in clinical trials, effectively inhibits growth of CTCL cells. The anti-cancer cell activity of 4SC-202 is however not limited to LSD1-inhibition, modulation of histone modifications, and consecutive alteration of gene expression. Indeed, the compound is also a potent microtubule-destabilizing agent.

Project description:Development of targeted therapy for hepatocellular carcinoma (HCC) remains a major challenge. We have recently identified an elevated expression of the fifth subunit of COP9 signalosome (CSN5) in early HCC as compared to dysplastic stage. In the present study, we explored the possibility of CSN5 being a potential therapeutic target for HCC. We demonstrate that CSN5 knockdown by small interfering (si) RNA caused a block in cell proliferation and cell cycle progression, and induced apoptosis of HCC cells in vitro. The transcriptomic analysis of CSN5 knockdown signature by microarray showed that the anti-proliferative effect was driven by a common subset of molecular alterations including downregulation of CDK6 or ITGB1, which are involved in cell cycle regulation or tumor invasion and functionally interconnected with key oncogenic regulators such as TGFβ1 and MYC. Western blot assay showed that CSN5 depletion significantly increases the phosphorylation of Smad2/3 and decreases the expression of ITGB1, CDK6 and cyclin D1 proteins, restoring the tumor suppressive function of RB and thereby contributing to inhibition of cell cycle progression. In addition, CSN5 knockdown increased the expression of Bak but decreased the level of Bcl-2, subsequently leading to apoptosis through the activation of caspase-3. A chemically modified variant of CSN5 siRNA was then selected for in vivo application based on the growth inhibitory effect and minimal induction of unwanted immune response. Systemic delivery of the CSN5 3/8 variant by stable-nucleic-acid-lipid-particles (SNALP) significantly suppressed the tumor growth in Huh7-luc+ orthotopic xenograft model. Taken together, these results indicate that CSN5 plays a pivotal role in HCC pathogenesis and maybe an attractive molecular target for systemic HCC therapy. Huh7 and HepG2 cells were treated with CSN5 siRNA and negative control siRNA for 48 hours and subjected to Illumina microarray analysis. 4 replicates each.

Dataset Information

Mechanism, Consequences, and Therapeutic Targeting of Abnormal IL15 Signaling in Cutaneous T-cell Lymphoma.

Publications

Mechanism, Consequences, and Therapeutic Targeting of Abnormal IL15 Signaling in Cutaneous T-cell Lymphoma.

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