Background: Cutaneous T cell lymphoma (CTCL) is a non-Hodgkin lymphoma characterized by proliferation of skin-homing T cells. STAT signaling plays an important role in the different CTCL subtypes, ...promoting carcinogenesis. The expression of STAT4, required for the T helper (Th) 1 differentiation, is firstly overexpressed in early stages of the disease and lost in later stages. In advanced stages, CTCL cells acquire a more Th2-like phenotype with concomitant high expression of STAT6. Resminostat, an epigenetic modulator with inhibitory potency against HDAC class I, lib and IV was investigated in vitro on its anti-tumoral efficacy against CTCL-derived cell lines and its impact on STAT4/STAT6 expression. Material and Methods: Mycosis fungoides (MF) and Sezary syndrome (SzS) are the most common and the leukemic form of CTCL, respectively Resminostat was tested for its effects on the viability of MyLa (MF) and HuT78 (SzS) cells. In order to evaluate the inhibitory effects, the primary effect, the hyperacetylation, as well as cell cycle distribution and apoptosis induction were determined. Further, resminostat's impact on the aberrantly activated STAT signaling in CTCL cells was investigated. Results: Resminostat exhibited a dose-dependent inhibition of the viability of MyLa and HuT78 cells. High-throughput bead-based ELISA revealed the hyperacetylation of various histone H3 residues upon treating the SzS cells with resminostat. Moreover, the epigenetic modulating small molecule induced apoptosis while only marginally affecting the cell cycle in these cell lines. Treatment of MyLa cells with resminostat reversed the anomalous STAT expression, namely inducing an increase in STAT4 and a decrease in STAT6 expression. Conclusion: Resminostat displayed conclusive in vitro anti-tumor activities both in MF and SzS cells. The regulation of the aberrant STAT signaling on transcription level suggests a stabilization of the less advanced CTCL stage (Th1) or even a reconversion of the advanced (Th2) to the less advanced Th1 phenotype. Hence, resminostat represents a promising treatment option for indolent as well as aggressive CTCL subtypes, possibly as maintenance therapy In upcoming functional assays, the impact of resminostat on the immunopathogenesis of CTCL will be analyzed and the anti-tumoral effects confirmed in an in vivo model.
Summary
Cardiac allograft vasculopathy (CAV) affects approximately 30% of cardiac transplant patients at 5 years post‐transplantation. To date, there are few CAV treatment or prevention options, none ...of which are highly effective. The aim of the study was to investigate the effect of thalidomide on the development of CAV. The effect of thalidomide treatment on chronic rejection was assessed in rat orthotopic aortic transplants in allogeneic F344 or syngeneic Lew rats (n = 6 per group). Animals were left untreated or received thalidomide for 30 days post‐transplant, and evidence of graft CAV was determined by histology (trichrome and immunohistochemistry) and intragraft cytokine measurements. Animals that received thalidomide treatment post‐transplant showed markedly reduced luminal obliteration, with concomitant rescue of smooth muscle cells (SMCs) in the aortic media of grafts. Thalidomide counteracted neointimal hyperplasia by preventing dedifferentiation of vascular SMCs. Measurement of intragraft cytokine levels after thalidomide treatment revealed downregulation of matrix metalloproteinase 8 and monocyte chemotactic protein 1, cytokines involved in tissue remodelling and inflammation, respectively. Importantly, no negative side effects of thalidomide were observed. Thalidomide treatment prevents CAV development in a rodent model and is therefore potentially useful in clinical applications to prevent post‐transplant heart rejection.
Bone marrow-derived mesenchymal stem cells (MSCs) are multipotent and secrete angiogenic factors, which could help patients with occlusive arterial diseases. We hypothesize that MSCs, in comparison ...to fibroblasts, survive better under hypoxic conditions in vitro and in vivo. MSCs and fibroblasts from L2G mice expressing firefly luciferase and GFP were cultured in normoxic and hypoxic conditions for 24 hours. In vitro cell viability was tested by detecting apoptosis and necrosis. MSCs released higher amounts of VEGF (281.1 +/- 62.6 pg/ml) under hypoxic conditions compared to normoxia (154.9 +/- 52.3 pg/ml, p = NS), but were less tolerant to hypoxia (45 +/- 7.9%) than fibroblasts (28.1 +/- 3.6%, p = NS). A hindlimb ischemia model was created by ligating the femoral artery of 18 FVB mice. After one week, 1 x 106 cells (MSCs, fibroblasts or saline) were injected into the limb muscles of each animal (n = 6 per group). Bioluminescence measurement to assess the viability of luciferase positive cells showed significant proliferation of MSCs on day four compared to fibroblasts (p = 0.001). Three weeks after cell delivery, the capillary to muscle fiber ratio of ischemic areas was analyzed. In the MSC group, vessel density was significantly higher than in the fibroblast or control group (0.5 +/- 0.08 and 0.3 +/- 0.03). Under hypoxia, MSCs produced more VEGF compared to normal conditions and MSC transplantation into murine ischemic limbs led to an increase in vessel density, although MSC survival was limited. This study suggests that MSC transplantation may be an effective and clinically relevant tool in the therapy of occlusive arterial diseases.
IntroductionCutaneous T cell lymphoma (CTCL) is a heterogeneous group of extra-nodal non-Hodgkin lymphomas arising from transformation and clonal expansion of skin-homing T cells. Epigenetics ...alterations have been described for the molecular pathogenesis of CTCL. Resminostat is a potent, orally bioavailable histone deacetylase (HDAC) inhibitor targeting class I, IIb and IV and is currently in phase II of clinical development. Resminostat showed anti-tumoral in vitro efficacy by inhibiting proliferation of CTCL cell lines. The aim of this study was to elucidate resminostat’s molecular mechanism of action in CTCL using a genome-wide approach.Material and methodsResminostat’s mechanism of action was analysed using a set of established CTCL cell lines representing different types of CTCL. Global lysine acetylation was evaluated by flow cytometry and specific histone H3 acetylation at lysine residue 27 (H3K27ac) was mapped genome-widely by chromatin immunoprecipitation followed by sequencing (ChIP-seq). Resminostat’s effect on global gene expression was analysed by RNA-seq focusing on CTCL-relevant pathways and genes.Results and discussionsResminostat increased total lysine acetylation in CTCL cell lines. The active epigenetic mark H3K27ac dose-dependently accumulated on a genome-wide level as detected by ChIP-seq analysis. Global gene expression profiling revealed both gene induction and repression by resminostat. First data showed a regulation of T helper (Th) 1 and 2 specific genes implying that resminostat normalises the Th1/Th2 imbalance in CTCL which is discussed to be associated with disease progression. Moreover, expression of skin-homing receptor genes was reduced by resminostat indicating a potential effect of resminostat on the cutaneous tropism of malignant T cells. Furthermore, the pruritus mediator IL-31 was downregulated by resminostat suggesting an attenuation of itching and thus indicating an important benefit for CTCL patients regarding their health-related quality of life.ConclusionFirst results from a genome-wide study suggest a promising impact of resminostat on different targets relevant for the pathogenesis of CTCL. These data broaden our understanding of resminostat’s molecular mechanism of action in CTCL and support our current clinical phase II trial evaluating resminostat for maintenance treatment of patients with advanced stage CTCL (RESMAIN, NCT02953301).
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