ABSTRACT
BACKGROUND: Photochemotherapy with psoralen and ultraviolet A (PUVA), with or without adjuvant interferon-α (IFN-α), is a first-line therapy for early-stage mycosis fungoides and other forms of cutaneous T-cell lymphoma (CTCL). However, the mechanism by which PUVA with IFN-α work in CTCL is poorly understood. PURPOSE: To develop a model to investigate the mechanisms of PUVA and PUVA with IFN-α in CTCL cells. METHODS: An in vitro model to study the molecular mechanisms of PUVA was created using two different CTCL cell lines, MyLa, which has functional p53, and HuT-78, in which p53 is inactivated due to a homozygous nonsense mutation. RESULTS: PUVA caused G2/M cell cycle block and apoptosis of MyLa and HuT-78 accompanied by increase in the expression of the mitochondrial pro-apoptotic genes Bax, BAK, and PUMA and a downregulation in anti-apoptotic Bcl-2. p53 was induced and c-Myc was repressed by PUVA, but neither were essential for PUVA-induced apoptosis. IFN-α augmented PUVA-induced apoptosis via the JAK1 pathway, and this activity could be inhibited by ruxolitinib. CONCLUSION: PUVA induces p53-independent apoptosis in CTCL cell lines, and this process is augmented by type I interferons via the JAK1 pathway.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Interferon-alpha/pharmacology , Lymphoma, T-Cell, Cutaneous/drug therapy , PUVA Therapy , Signal Transduction/drug effects , Apoptosis/radiation effects , Apoptosis Regulatory Proteins/genetics , Cell Line, Tumor , G2 Phase Cell Cycle Checkpoints/drug effects , G2 Phase Cell Cycle Checkpoints/radiation effects , Gene Expression , Humans , Janus Kinase 1/metabolism , Janus Kinase Inhibitors/pharmacology , Lymphoma, T-Cell, Cutaneous/genetics , Lymphoma, T-Cell, Cutaneous/metabolism , M Phase Cell Cycle Checkpoints/radiation effects , Nitriles , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-myc/metabolism , Pyrazoles/pharmacology , Pyrimidines , STAT1 Transcription Factor/metabolism , Signal Transduction/radiation effects , Tumor Suppressor Protein p53/genetics , bcl-2 Homologous Antagonist-Killer Protein/genetics , bcl-2-Associated X Protein/geneticsABSTRACT
BACKGROUND: Psoralen plus ultraviolet A (PUVA) photochemotherapy is a combination treatment used for inflammatory and neoplastic skin diseases such as mycosis fungoides (MF), the most common type of cutaneous T-cell lymphoma (CTCL). However, 30% of MF patients do not respond sufficiently to PUVA and require more aggressive therapies. OBJECTIVE: The aim of this project was to investigate whether inhibition of Ataxia Telangiectasia and Rad3 related kinase (ATR) may enhance efficacy of phototherapy. METHODS: CTCL cell lines (MyLa2000, SeAx and Mac2a) served as in vitro cell models. ATR and Chk1 were inhibited by small molecule antagonists VE-821, VE-822 or Chir-124, or by small interfering RNAs (siRNAs). Cell cycle and viability were assessed by flow cytometry. RESULTS: Small molecule inhibitors of ATR and Chk1 potently sensitized all cell lines to PUVA and, importantly, also to UVA, which by itself did not cause apoptotic response. VE-821/2 blocked ATR pathway activation and released the cells from the G2/M block caused by UVA and PUVA, but did not affect apoptosis caused by other chemotherapeutics (etoposide, gemcitabine, doxorubicine) or by hydrogen peroxide. Knockdown of ATR and Chk1 with siRNA also blocked the ATR pathway and released the cells from G2/M block but did not sensitize the cells to UVA as observed with the small molecule inhibitors. The latter suggested that the synergism between VE-821/2 or Chir-124 and UVA was not solely caused by specific blocking of ATR kinase but also ATR-independent photosensitization. This hypothesis was further verified by administrating VE-821/2 or Chir-124 before and after UVA irradiation, as well as comparing their activity with other ATR and Chk1 inhibitors (AZD6738 and MK8776). We found that only VE-821/2 and Chir-124 kinase inhibitors had synergistic effect with UVA, and only if applied before treatment with UVA. CONCLUSION: Small molecule ATR and Chk1 inhibitors potently sensitize lymphoma cells to UVA radiation and induce a prominent apoptotic response. Interestingly, this effect is due to the dual (kinase inhibiting and photosensitizing) mode of action of these compounds.
Subject(s)
Checkpoint Kinase 1/antagonists & inhibitors , Lymphoma, T-Cell, Cutaneous/pathology , Photosensitizing Agents/pharmacology , Ultraviolet Rays , Ataxia Telangiectasia Mutated Proteins/antagonists & inhibitors , Cell Cycle , Cell Line, Tumor , Cell Proliferation , Cell Survival , Flow Cytometry , Histones/metabolism , Humans , Isoxazoles/pharmacology , Lymphoma, T-Cell, Cutaneous/metabolism , Lymphoma, T-Cell, Cutaneous/therapy , Phosphorylation , Photochemotherapy , Pyrazines/pharmacology , Quinolines/pharmacology , Quinuclidines/pharmacology , RNA, Small Interfering/metabolism , Sulfones/pharmacology , Tumor Cells, CulturedABSTRACT
PURPOSE: Sustained inflammation is a key feature of mycosis fungoides (MF), the most common form of cutaneous T-cell lymphoma (CTCL). Resident IL9-producing T cells have been found in skin infections and certain inflammatory skin diseases, but their role in MF is currently unknown. EXPERIMENTAL DESIGN: We analyzed lesional skin from patients with MF for the expression of IL9 and its regulators. To determine which cells were producing IL9, high-throughput sequencing was used to identify malignant clones and Vb-specific antibodies were employed to visualize malignant cells in histologic preparations. To explore the mechanism of IL9 secretion, we knocked down STAT3/5 and IRF4 by siRNA transfection in CTCL cell lines receiving psoralen+UVA (PUVA) ± anti-IL9 antibody. To further examine the role of IL9 in tumor development, the EL-4 T-cell lymphoma model was used in C57BL/6 mice. RESULTS: Malignant and reactive T cells produce IL9 in lesional skin. Expression of the Th9 transcription factor IRF4 in malignant cells was heterogeneous, whereas reactive T cells expressed it uniformly. PUVA or UVB phototherapy diminished the frequencies of IL9- and IL9r-positive cells, as well as STAT3/5a and IRF4 expression in lesional skin. IL9 production was regulated by STAT3/5 and silencing of STAT5 or blockade of IL9 with neutralizing antibodies potentiated cell death after PUVA treatment in vitro IL9-depleted mice exhibited a reduction of tumor growth, higher frequencies of regulatory T cells, and activated CD4 and CD8 T lymphocytes. CONCLUSIONS: Our results suggest that IL9 and its regulators are promising new targets for therapy development in mycosis fungoides. Clin Cancer Res; 22(13); 3328-39. ©2016 AACR.
