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Immunotherapy has achieved tremendous success in melanoma. However, only around 50% of advanced melanoma patients benefit from immunotherapy. Cyclin-dependent kinase inhibitor 2A (CDKN2A), encoding the two tumor-suppressor proteins p14 ARF and p16 INK4a , belongs to the most frequently inactivated gene loci in melanoma and leads to decreased T cell infiltration. While the role of p16 INK4a has been extensively investigated, knowledge about p14 ARF in melanoma is scarce. In this study, we elucidate the impact of reduced p14 ARF expression on melanoma immunogenicity. Knockdown of p14 ARF in melanoma cell lines diminished their recognition and killing by melanoma differentiation antigen (MDA)-specific T cells. Resistance was caused by a reduction of the peptide surface density of presented MDAs. Immunopeptidomic analyses revealed that antigen presentation via human leukocyte antigen class I (HLA-I) molecules was enhanced upon p14 ARF downregulation in general, but absolute and relative expression of cognate peptides was decreased. However, this phenotype is associated with a favorable outcome for melanoma patients. Limiting Wnt5a signaling reverted this phenotype, suggesting an involvement of non-canonical Wnt signaling. Taken together, our data indicate a new mechanism limiting MDA-specific T cell responses by decreasing both absolute and relative MDA-peptide presentation in melanoma., (© 2024 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Elevated levels of peripheral blood and tumor tissue neutrophils are associated with poorer clinical response and therapy resistance in melanoma. The underlying mechanism and the role of neutrophils in targeted therapy is still not fully understood. Serum samples of patients with advanced melanoma were collected and neutrophil-associated serum markers were measured and correlated with response to targeted therapy. Blood neutrophils from healthy donors and patients with advanced melanoma were isolated, and their phenotypes, as well as their in vitro functions, were compared. In vitro functional tests were conducted through nonadherent cocultures with melanoma cells. Protection of melanoma cell lines by neutrophils was assessed under MAPK inhibition. Blood neutrophils from advanced melanoma patients exhibited lower CD16 expression compared to healthy donors. In vitro, both healthy-donor- and patient-derived neutrophils prevented melanoma cell apoptosis upon dual MAPK inhibition. The effect depended on cell-cell contact and melanoma cell susceptibility to treatment. Interference with protease activity of neutrophils prevented melanoma cell protection during treatment in cocultures. The negative correlation between neutrophils and melanoma outcomes seems to be linked to a protumoral function of neutrophils. In vitro, neutrophils exert a direct protective effect on melanoma cells during dual MAPK inhibition. This study further hints at a crucial role of neutrophil-related protease activity in protection.

Background: The mitogen-activated protein kinase (MAPK) signaling pathway is frequently hyperactivated in malignant melanoma and its inhibition has proved to be an efficient treatment option for cases harboring BRAF V600 mutations (BRAF Mut ). However, there is still a significant need for effective targeted therapies for patients with other melanoma subgroups characterized by constitutive MAPK activation, such as tumors with NRAS or NF-1 alterations (NRAS Mut , NF-1 LOF ), as well as for patients with MAPK pathway inhibitor-resistant BRAF Mut melanomas, which commonly exhibit a reactivation of this pathway. p90 ribosomal S6 kinases (RSKs) represent central effectors of MAPK signaling, regulating cell cycle progression and survival., Methods: RSK activity and the functional effects of its inhibition by specific small molecule inhibitors were investigated in established melanoma cell lines and patient-derived short-term cultures from different MAPK pathway-hyperactivated genomic subgroups (NRAS Mut , BRAF Mut , NF-1 LOF ). Real-time qPCR, immunoblots and flow cytometric cell surface staining were used to explore the molecular changes following RSK inhibition. The effect on melanoma cell growth was evaluated by various two- and three-dimensional in vitro assays as well as with melanoma xenograft mouse models. Co-cultures with gp100- or Melan-A-specific cytotoxic T cells were used to assess immunogenicity of melanoma cells and associated T-cell responses., Results: In line with elevated activity of the MAPK/RSK signaling axis, growth and survival of not only BRAF Mut but also NRAS Mut and NF-1 LOF melanoma cells were significantly impaired by RSK inhibitors. Intriguingly, RSK inhibition was particularly effective in three-dimensional growth settings with long-term chronic drug exposure and suppressed tumor cell growth of in vivo melanoma models. Additionally, our study revealed that RSK inhibition simultaneously promoted differentiation and immunogenicity of the tumor cells leading to enhanced T-cell activation and melanoma cell killing., Conclusions: Collectively, RSK inhibitors exhibited both multi-layered anti-tumor efficacy and broad applicability across different genomic melanoma subgroups. RSK inhibition may therefore represent a promising novel therapeutic strategy for malignant melanoma with hyperactivated MAPK signaling., (© 2023. The Author(s).)

Background: Eosinophils appear to contribute to the efficacy of immunotherapy and their frequency was suggested as a predictive biomarker. Whether this observation could be transferred to patients treated with targeted therapy remains unknown., Methods: Blood and serum samples of healthy controls and 216 patients with advanced melanoma were prospectively and retrospectively collected. Freshly isolated eosinophils were phenotypically characterized by flow cytometry and co-cultured in vitro with melanoma cells to assess cytotoxicity. Soluble serum markers and peripheral blood counts were used for correlative studies., Results: Eosinophil-mediated cytotoxicity towards melanoma cells, as well as phenotypic characteristics, were similar when comparing healthy donors and patients. However, high relative pre-treatment eosinophil counts were significantly associated with response to MAPKi ( p = 0.013). Eosinophil-mediated cytotoxicity towards melanoma cells is dose-dependent and requires proximity of eosinophils and their target in vitro. Treatment with targeted therapy in the presence of eosinophils results in an additive tumoricidal effect. Additionally, melanoma cells affected eosinophil phenotype upon co-culture., Conclusion: High pre-treatment eosinophil counts in advanced melanoma patients were associated with a significantly improved response to MAPKi. Functionally, eosinophils show potent cytotoxicity towards melanoma cells, which can be reinforced by MAPKi. Further studies are needed to unravel the molecular mechanisms of our observations.

Background: Merkel cell carcinoma (MCC) is an aggressive skin cancer in which PD-1/PD-L1 blockade has shown remarkable response rates. However, a significant proportion of patients shows primary or secondary resistance against PD-1/PD-L1 inhibition, with HLA class-I downregulation and insufficient influx of CD8 + T cells into the tumor as possible immune escape mechanisms. Histone deacetylase inhibitors (HDACi) have been demonstrated to reverse low HLA class-I expression caused by epigenetic downregulation of the antigen machinery (APM) in vitro and in pre-clinical models in vivo., Case Presentations: We report four cases of patients with metastatic MCC who did not respond to immunotherapy by PD-1/PD-L1 blockade. Two of the patients received, subsequently, the HDACi panobinostat in combination with PD-1/PD-L1 blockade. Tumor biopsies of the patients were analyzed for cellular and molecular markers of antigen processing and presentation as well as the degree of T-cell infiltration., Results and Conclusion: Low expression of APM-related genes associated with low HLA class-I surface expression was observed in all MCC patients, progressing on PD-1/PD-L1 blockade. In one evaluable patient, of the two treated with the combination therapy of the HDACi, panobinostat and PD-1/PD-L1 blockade, reintroduction of HLA class-I-related genes, enhanced HLA class-I surface expression, and elevated CD8 + T-cell infiltration into the MCC tumor tissue were observed; however, these changes did not translate into a clinical benefit. Our findings suggest that HDACi may be useful to overcome HLA class-I downregulation as a resistance mechanism against anti-PD-1/PD-L1 antibodies in MCC patients. Prospective clinical trials are needed to evaluate this notion.

Chromium allergy is a common occupational skin disease mediated by chromium (VI)-specific T cells that induce delayed-type hypersensitivity in sensitized individuals. Additionally, chromium (VI) can act as an irritant. Both responses critically require innate immune activation, but if and how chromium (VI) elicits this signal is currently unclear. Using human monocytes, primary human keratinocytes, and murine dendritic cells we show that chromium (VI) compounds fail to trigger direct proinflammatory activation but potently induce processing and secretion of IL-1β. IL-1β release required priming by phorbol-ester or toll-like receptor stimulation and was prevented by inhibition of K + efflux, NLRP3 depletion or caspase-1 inhibition, identifying chromium (VI) as a hapten activator of the NLRP3 inflammasome. Inflammasome activation was initiated by mitochondrial reactive oxygen species production triggered by chromium (VI), as indicated by sensitivity to treatment with the ROS scavenger N-acetyl cysteine and a coinciding failure of K + efflux, caspase-1, or NLRP3 inhibition to prevent mitochondrial reactive oxygen species accumulation. IL-1β release further correlated with cytotoxicity that was secondary to reactive oxygen species, K + efflux, and NLRP3 activation. Trivalent chromium was unable to induce mitochondrial reactive oxygen species production, inflammasome activation, and cytotoxicity, suggesting that oxidation state-specific differences in mitochondrial reactivity may determine inflammasome activation and allergic/irritant capacity of different chromium compounds., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: Low dose hyperradiosensitivity (HRS) has been observed in HGL21- and T98G human glioblastoma cells in vitro. The present study investigates whether these effects translate into improved outcome of ultrafractionated irradiation (UF) in vivo., Material and Methods: T98G or HGL21 were transplanted on the hind leg of nude mice. Tumours were irradiated with UF (3 fractions of 0.4 Gy per day, interval 4 h, 7 days per week) or with conventional fractionation (CF; 1 fraction of 1.68 Gy per day, 5 days per week) over 2 or 4 weeks in HGL21 and 2,4 or 6 weeks in T98G. In HGL21, graded top-up doses under clamped hypoxia were applied after 4 weeks of fractionated irradiation. Additional groups of animals were irradiated with single doses under clamp hypoxic conditions with or without whole body irradiation (WBI) before tumour transplantation. Experimental endpoints were growth delay (time to 5-fold starting volume, GD(V5)) and local tumour control., Results: In T98G tumours median relative GD(V5) was 1.2 [95% C.I. 0.96; 8] in the CF and 0.8 [0.7; 1.02] in the UF arm (p = 0.009) indicating that ultrafractionation is less efficient than conventional fractionation. The TCD50 value of 33.5 Gy [22; 45] after UF was higher than TCD50 of 23.6 Gy [16; 31] after CF (p = 0.15). In HGL21 the median relative GD(V5) was not significantly different between CF and UF. The top-up TCD50 value of 16.1 Gy [95% C.I. 9; 23 Gy] after CF was significantly lower than the corresponding value of 33.2 Gy [23; 44] after UF irradiation (p = 0.007), indicating a higher efficacy of CF compared to UF., Conclusion: The results on human T98G and HGL21 glioblastoma do not support the hypothesis that HRS in vitro translates into improved outcome of ultrafractionated irradiation in vivo.

Background and Purpose: Low-dose hyperradiosensitivity (HRS), i.e., a relatively higher efficacy of doses < or = 0.5 Gy compared to doses > 1 Gy, has been shown in a number of tumor cell lines in vitro. Therefore ultrafractionated irradiation, i.e., application of very low doses per fraction, has been proposed to improve the effects of radiotherapy. The present study investigates ultrafractionation (UF) in radioresistant murine DDL1 T-cell lymphoma in mice., Material and Methods: UF was performed with 0.4 Gy per fraction, three fractions per day at 7 days per week, and conventional fractionation (CF) with 1.68 Gy per fraction, one fraction per day at 5 days per week. Tumor growth delay was evaluated for 2, 4 and 6 weeks of irradiation as time that tumors needed to reach fivefold the starting volume (GD(V5))., Results: GD(V5) was not significantly different between UF and CF. The composite median relative GD(V5) calculated for all tumors irradiated in the present study was 1.00 [95% confidence interval 0.99; 1.08] in the CF and 0.99 [0.92; 1.01] in the UF arm (p = 0.24)., Conclusion: UF was not more efficient than CF in DDL1 tumors. Taken together with previous experiments on human A7 glioblastoma, which showed a negative effect of UF on local tumor control, the preclinical data obtained in this laboratory so far do not support the use of ultrafractionated schedules in radiotherapy.

Intracellular potentials were measured in a SV-40 virus-transformed cell clone derived from human nonpigmented ciliary epithelium using the microelectrode technique. (1) Membrane potential averaged -50.2 mV (+/- 0.6, n = 207). (2) Increasing the extracellular K+ concentration depolarized the membrane voltage. The amplitude of this potential response was reduced in the presence of 1 mM Ba2+. (3) Superfusing the cells with a Ca2+-free solution containing 1 mM EGTA depolarized the intracellular potential and diminished the voltage response upon increasing extracellular K+. (4) Extracellular alkalinization hyperpolarized the membrane potential and increased the voltage amplitude on increasing extracellular K+. (5) Addition of ouabain immediately reduced the intracellular potential. Removing extracellular K+ depolarized membrane voltage, readdition of K+ after K+ depletion transiently hyperpolarized intracellular voltage. Both potential responses were inhibited in the presence of ouabain. (6) Replacing extracellular Cl- by cyclamate resulted in a transient depolarization followed by a hyperpolarization. In the presence of SITS or DIDS (greater than or equal to 0.1 mM) the electrical responses of the cell membrane to Cl- replacement were blocked. We conclude that cultured human nonpigmented ciliary epithelial cells possess an electrogenic Na+/K+-ATPase, a K+ conductance modulated by Ca2+ and pH, and a Cl- conductance sensitive to stilbene derivatives.

Human nonpigmented ciliary epithelial cells (NPE) were grown in tissue culture after transformation with an origin-defective mutant of SV-40 DNA. In these cells membrane potentials (V) were measured using the microelectrode technique. Addition of 10(-4) M acetylcholine led to a bisphasic voltage response. An immediate, transient hyperpolarization was followed by a sustained depolarization below the steady state level. These responses were irreversibly blocked by 10(-5) M atropine. In Ca2+-free media the initial addition of acetylcholine resulted in an unchanged voltage response. A second application of acetylcholine in Ca2+-free solution evoked only an abortive response of V, and further addition had no effect on V. In the presence of Ca2+ channel blockers (10(-5) M verapamil, 1 mM Co2+) the acetylcholine-induced response of the membrane potential was not changed. The initial hyperpolarization induced by acetylcholine was reduced by 33 +/- 3% (n = 6) in the presence of 2 mM Ba2+ and by 79 +/- 6% (n = 6) in the presence of 1 mM quinidine. Moreover, the amplitude of the hyperpolarization was dependent on the extracellular K+ concentration. With increasing extracellular K+ concentration (and decreasing transmembrane K+ gradient) the acetylcholine-induced hyperpolarization was reduced. To further elucidate the role of Ca2+ in the acetylcholine-induced responses, we measured cytoplasmic Ca2+ activity using the fluorescence of intracellularly trapped Fura-2. Cytoplasmic Ca2+ activity increased immediately and transiently upon addition of acetylcholine. We conclude that acetylcholine transiently hyperpolarizes V in cultured human NPE by activation of K+ channels mediated by mobilization of Ca2+ from intracellular stores.

Bovine non-pigmented ciliary epithelial cells (NPE) have been isolated by a technique of selective adhesion to tissue culture plastic. NPE cells in primary culture proliferated and maintained epithelial-like morphology for about 4 weeks in tissue culture medium containing 10% fetal calf serum. If grown for longer than 4 weeks in serum-containing medium, cells changed their morphology and became elongated and spindle-shaped. Membrane potentials were measured using conventional microelectrodes. In NPE cells of epithelial-like shape, replacing extracellular Na+ induced a transient hyperpolarization of the membrane potential, while in elongated cells of spindle-shaped morphology an immediate depolarization was observed. We therefore only used epithelial-like NPE for further experiments. In these cells the mean membrane potential was -40.3 +/- 0.5 mV (n = 36). Relative K+ conductance was increased by extracellular alkalinization. Removing extracellular K+ led to a depolarization and readdition of K+ to K+ depleted cells resulted in a hyperpolarization. Both voltage responses were sensitive to ouabain, indicating that Na+/K+ ATPase is inhibited by K+ replacement, and that there is overshoot-activation of the pump when K+ is readded. Extracellular Cl- replacement led to a DIDS sensitive, transient depolarization, which is compatible with a stilbene-sensitive Cl(-)-conductance. Removing HCO3- led to a Na+ dependent and DIDS-sensitive depolarization. However, the electrical response on replacement of extracellular Na+ was not influenced by DIDS or the extracellular HCO3(-)-concentration.