Augmenting MEK inhibitor efficacy in BRAF wild-type melanoma: synergistic effects of disulfiram combination therapy.

BACKGROUND: MEK inhibitors (MEKi) were shown to be clinically insufficiently effective in patients suffering from BRAF wild-type (BRAF WT) melanoma, even if the MAPK pathway was constitutively activated due to mutations in NRAS or NF-1. Thus, novel combinations are needed to increase the efficacy and duration of response to MEKi in BRAF WT melanoma. Disulfiram and its metabolite diethyldithiocarbamate are known to have antitumor effects related to cellular stress, and induction of endoplasmic reticulum (ER) stress was found to synergize with MEK inhibitors in NRAS-mutated melanoma cells. Therefore, we investigated the combination of both therapeutics to test their effects on BRAF-WT melanoma cells and compared them with monotherapy using the MEKi trametinib. METHODS: The effects of combined therapy with disulfiram or its metabolite diethyldithiocarbamate and the MEKi trametinib were evaluated in a series of BRAF-WT melanoma cell lines by measuring cell viability and apoptosis induction. Cytotoxicity was additionally assessed in 3D spheroids, ex vivo melanoma slice cultures, and in vivo xenograft mouse models. The response of melanoma cells to treatment was studied at the RNA and protein levels to decipher the mode of action. Intracellular and intratumoral copper measurements were performed to investigate the role of copper ions in the antitumor cytotoxicity of disulfiram and its combination with the MEKi. RESULTS: Diethyldithiocarbamate enhanced trametinib-induced cytotoxicity and apoptosis induction in 2D and 3D melanoma culture models. Mechanistically, copper-dependent induction of oxidative stress and ER stress led to Janus kinase (JNK)-mediated apoptosis in melanoma cells. This mechanism was also detectable in patient-derived xenograft melanoma models and resulted in a significantly improved therapeutic effect compared to monotherapy with the MEKi trametinib. CONCLUSIONS: Disulfiram and its metabolite represent an attractive pharmaceutical approach to induce ER stress in melanoma cells that potentiates the antitumor effect of MEK inhibition and may be an interesting candidate for combination therapy of BRAF WT melanoma.

BRAF and MEK inhibitor combinations induce potent molecular and immunological effects in NRAS-mutant melanoma cells: Insights into mode of action and resistance mechanisms.

About 25% of melanoma harbor activating NRAS mutations, which are associated with aggressive disease therefore requiring a rapid antitumor intervention. However, no efficient targeted therapy options are currently available for patients with NRAS-mutant melanoma. MEK inhibitors (MEKi) appear to display a moderate antitumor activity and also immunological effects in NRAS-mutant melanoma, providing an ideal backbone for combination treatments. In our study, the MEKi binimetinib, cobimetinib and trametinib combined with the BRAF inhibitors (BRAFi) encorafenib, vemurafenib and dabrafenib were investigated for their ability to inhibit proliferation, induce apoptosis and alter the expression of immune modulatory molecules in sensitive NRAS-mutant melanoma cells using two- and three-dimensional cell culture models as well as RNA sequencing analyses. Furthermore, NRAS-mutant melanoma cells resistant to the three BRAFi/MEKi combinations were established to characterize the mechanisms contributing to their resistance. All BRAFi induced a stress response in the sensitive NRAS-mutant melanoma cells thereby significantly enhancing the antiproliferative and proapoptotic activity of the MEKi analyzed. Furthermore, BRAFi/MEKi combinations upregulated immune relevant molecules, such as ICOS-L, components of antigen-presenting machinery and the "don't eat me signal" molecule CD47 in the melanoma cells. The BRAFi/MEKi-resistant, NRAS-mutant melanoma cells counteracted the molecular and immunological effects of BRAFi/MEKi by upregulating downstream mitogen-activated protein kinase pathway molecules, inhibiting apoptosis and promoting immune escape mechanisms. Together, our study reveals potent molecular and immunological effects of BRAFi/MEKi in sensitive NRAS-mutant melanoma cells that may be exploited in new combinational treatment strategies for patients with NRAS-mutant melanoma.

Exploring the In Vitro and In Vivo Therapeutic Potential of BRAF and MEK Inhibitor Combination in NRAS-Mutated Melanoma.

INTRODUCTION: Patients with NRAS-mutant metastatic melanoma often have an aggressive disease requiring a fast-acting, effective therapy. The MEK inhibitor binimetinib shows an overall response rate of 15% in patients with NRAS-mutant melanoma, providing a backbone for combination strategies. Our previous studies demonstrated that in NRAS-mutant melanoma, the antitumor activity of the MEK inhibitor binimetinib was significantly potentiated by the BRAFV600E/K inhibitor encorafenib through the induction of ER stress, leading to melanoma cell death by apoptotic mechanisms. Encorafenib combined with binimetinib was well tolerated in a phase III trial showing potent antitumor activity in BRAF-mutant melanoma, making a rapid evaluation in NRAS-mutant melanoma imminently feasible. These data provide a mechanistic rationale for the evaluation of binimetinib combined with encorafenib in preclinical and clinical studies on NRAS-mutant metastatic melanoma. METHODS: The combination of BRAFi plus MEKi was tested in a monolayer culture of patient-derived cell lines and in corresponding patient-derived tissue slice cultures of NRAS-mutant melanoma. To investigate the treatment in vivo, NSG (NOD. Cg-PrkdcscidIl2rgtm1Wjl/SzJ) mice were subcutaneously injected with three different BRAF wild-type melanoma models harboring oncogenic NRAS mutations and treated orally with encorafenib (6 mg/kg body weight, daily) with or without binimetinib (8 mg/kg body weight, twice daily). In parallel, an individual healing attempt was carried out by treating one patient with an NRAS-mutated tumor. RESULTS: Encorafenib was able to enhance the inhibitory effect on cell growth of binimetinib only in the cell line SKMel147 in vitro. It failed to enhance the apoptotic effect found in two other NRAS-mutated cell lines. Encorafenib led to a hyperactivation of ERK which could be reduced with the combinational treatment. In two of the three patient-derived tissue slice culture models of NRAS-mutant melanomas, a slight tendency of a combinatorial effect was seen which was not significant. Encorafenib showed a slight induction of the ER stress genes ATF4, CHOP, and NUPR1. The combinational treatment was able to enhance this effect, but not significantly. In the mouse model, the combination therapy of encorafenib with binimetinib resulted in reduced tumor growth compared to the control and encorafenib groups; however, the best effect in terms of tumor growth inhibition was measured in the binimetinib therapy group. The therapy showed no effect in an individual healing attempt for a patient suffering from metastatic, therapy-refractory NRAS-mutated melanoma. CONCLUSION: In in vitro and ex vivo settings, the combination therapy was observed to elicit a response; however, it did not amplify the efficacy observed with binimetinib alone, whereas in a patient, the combinational treatment remained ineffective. The preclinical in vivo data showed no increased combinatorial effect. However, the in vivo effect of binimetinib as monotherapy was unexpectedly high in the tested regimen. Nevertheless, binimetinib proved to be advantageous in the treatment of melanoma in vivo and led to high rates of apoptosis in vitro; hence, it still seems to be a good base for combination with other substances in the treatment of patients with NRAS-mutant melanoma.

PARP Inhibitors Effectively Reduce MAPK Inhibitor Resistant Melanoma Cell Growth and Synergize with MAPK Inhibitors through a Synthetic Lethal Interaction In Vitro and In Vivo.

The efficacy of targeting the MAPK signaling pathway in patients with melanoma is limited by the rapid development of resistance mechanisms that result in disease relapse. In this article, we focus on targeting the DNA repair pathway as an antimelanoma therapy, especially in MAPK inhibitor resistant melanoma cells using PARP inhibitors. We found that MAPK inhibitor resistant melanoma cells are particularly sensitive to PARP inhibitor treatment due to a lower basal expression of the DNA damage sensor ataxia-telangiectasia mutated (ATM). As a consequence, MAPK inhibitor resistant melanoma cells have decreased homologous recombination repair activity leading to a reduced repair of double-strand breaks caused by the PARP inhibitors. We validated the clinical relevance of our findings by ATM expression analysis in biopsies from patients with melanoma before and after development of resistance to MAPK inhibitors. Furthermore, we show that inhibition of the MAPK pathway induces a homologous recombination repair deficient phenotype in melanoma cells irrespective of their MAPK inhibitor sensitivity status. MAPK inhibition results in a synthetic lethal interaction of a combinatorial treatment with PARP inhibitors, which significantly reduces melanoma cell growth in vitro and in vivo. In conclusion, this study shows that PARP inhibitor treatment is a valuable therapy option for patients with melanoma, either as a single treatment or as a combination with MAPK inhibitors depending on ATM expression. Significance: We show that MAPK inhibitor resistant melanoma cells exhibit low ATM expression increasing their sensitivity toward PARP inhibitors and that a combination of MAPK/PARP inhibitors act synthetically lethal in melanoma cells. Our study shows that PARP inhibitor treatment is a valuable therapy option for patients with melanoma, either as a single treatment or as a combination with MAPK inhibitors depending on ATM expression, which could serve as a novel biomarker for treatment response.

Inhibition of p90 ribosomal S6 kinases disrupts melanoma cell growth and immune evasion.

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 BRAFV600 mutations (BRAFMut). 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 (NRASMut, NF-1LOF), as well as for patients with MAPK pathway inhibitor-resistant BRAFMut 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 (NRASMut, BRAFMut, NF-1LOF). 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 BRAFMut but also NRASMut and NF-1LOF 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.

Deep learning-based scoring of tumour-infiltrating lymphocytes is prognostic in primary melanoma and predictive to PD-1 checkpoint inhibition in melanoma metastases.

BACKGROUND: Recent advances in digital pathology have enabled accurate and standardised enumeration of tumour-infiltrating lymphocytes (TILs). Here, we aim to evaluate TILs as a percentage electronic TIL score (eTILs) and investigate its prognostic and predictive relevance in cutaneous melanoma. METHODS: We included stage I to IV cutaneous melanoma patients and used hematoxylin-eosin-stained slides for TIL analysis. We assessed eTILs as a continuous and categorical variable using the published cut-off of 16.6% and applied Cox regression models to evaluate associations of eTILs with relapse-free, distant metastasis-free, and overall survival. We compared eTILs of the primaries with matched metastasis. Moreover, we assessed the predictive relevance of eTILs in therapy-naïve metastases according to the first-line therapy. FINDINGS: We analysed 321 primary cutaneous melanomas and 191 metastatic samples. In simple Cox regression, tumour thickness (p < 0.0001), presence of ulceration (p = 0.0001) and eTILs ≤16.6% (p = 0.0012) were found to be significant unfavourable prognostic factors for RFS. In multiple Cox regression, eTILs ≤16.6% (p = 0.0161) remained significant and downgraded the current staging. Lower eTILs in the primary tissue was associated with unfavourable relapse-free (p = 0.0014) and distant metastasis-free survival (p = 0.0056). In multiple Cox regression adjusted for tumour thickness and ulceration, eTILs as continuous remained significant (p = 0.019). When comparing TILs in primary tissue and corresponding metastasis of the same patient, eTILs in metastases was lower than in primary melanomas (p < 0.0001). In therapy-naïve metastases, an eTILs >12.2% was associated with longer progression-free survival (p = 0.037) and melanoma-specific survival (p = 0.0038) in patients treated with anti-PD-1-based immunotherapy. In multiple Cox regression, lactate dehydrogenase (p < 0.0001) and eTILs ≤12.2% (p = 0.0130) were significantly associated with unfavourable melanoma-specific survival. INTERPRETATION: Assessment of TILs is prognostic in primary melanoma samples, and the eTILs complements staging. In therapy-naïve metastases, eTILs ≤12.2% is predictive of unfavourable survival outcomes in patients receiving anti-PD-1-based therapy. FUNDING: See a detailed list of funding bodies in the Acknowledgements section at the end of the manuscript.

Features and Long-Term Outcomes of Stage IV Melanoma Patients Achieving Complete Response Under Anti-PD-1-Based Immunotherapy.

BACKGROUND: Immune checkpoint inhibition (ICI) has changed the melanoma treatment spectrum. Few studies have examined the characteristics and long-term outcomes of patients achieving complete response (CR) under ICI. MATERIALS AND METHODS: We evaluated patients with unresectable stage IV melanoma treated with first-line ICI. The characteristics of those achieving CR were compared with those not achieving CR. Progression-free survival (PFS) and overall survival (OS) were assessed. Late-onset toxicities, response to second-line treatment, the prognostic value of clinicopathologic features, and blood markers were examined. RESULTS: A total of 265 patients were included; 41 (15.5%) achieved CR, while 224 (84.5%) had progressive disease, stable disease, or partial response. At the therapy start, those who had CR were more likely to be older than 65 years of age (p = 0.013), have a platelet-to-lymphocyte ratio below 213 (p = 0.036), and have lower lactate dehydrogenase levels (p = 0.008) than those not achieving a CR. For those who discontinued therapy after CR, the median follow-up time after CR was 56 months (interquartile range [IQR] 52-58) and the median time from CR to therapy end was 10 months (IQR 1-17). Five-year PFS after CR was 79% and 5-year OS was 83%. Most complete responders had a normalization of S100 at the time of CR (p < 0.001). In simple Cox regression analysis, age below 77 years at CR (p = 0.04) was associated with better prognosis after CR. Eight patients received second-line ICI; disease control was seen in 63%. Late immune-related toxicities occurred in 25% of patients, most being cutaneous immune-related toxicities. CONCLUSIONS: Response, according to the Response Evaluation Criteria in Solid Tumors (RECIST) criteria, is, until now, the most important prognostic factor, and CR is a valid surrogate marker for long-term survival in patients treated with ICI. Our results highlight the importance of investigating the optimal therapy duration in complete responders.

Relevant Membrane Transport Proteins as Possible Gatekeepers for Effective Pharmacological Ascorbate Treatment in Cancer.

Despite the increasing number of newly diagnosed malignancies worldwide, therapeutic options for some tumor diseases are unfortunately still limited. Interestingly, preclinical but also some clinical data suggest that the administration of pharmacological ascorbate seems to respond well, especially in some aggressively growing tumor entities. The membrane transport and channel proteins are highly relevant for the use of pharmacological ascorbate in cancer therapy and are involved in the transfer of active substances such as ascorbate, hydrogen peroxide, and iron that predominantly must enter malignant cells to induce antiproliferative effects and especially ferroptosis. In this review, the relevant conveying proteins from cellular surfaces are presented as an integral part of the efficacy of pharmacological ascorbate, considering the already known genetic and functional features in tumor tissues. Accordingly, candidates for diagnostic markers and therapeutic targets are mentioned.

Identification of stage I/II melanoma patients at high risk for recurrence using a model combining clinicopathologic factors with gene expression profiling (CP-GEP).

PURPOSE: Patients with cutaneous melanoma stage I/IIA disease are currently not eligible for adjuvant therapy, despite their risk for relapses and death. This study validates the ability of a model combining clinicopathologic factors with gene expression profiling (CP-GEP) to identify patients at high risk for disease recurrence in stage I/II and subgroup stage I/IIA. PATIENTS AND METHODS: 543 patients with stage I/II primary cutaneous melanoma from the University of Tuebingen diagnosed between 2000 and 2017 were analysed. All patients received sentinel lymph node biopsy (SLNB). Analysis was conducted for a separate group of 80 patients who did not undergo SLNB. RESULTS: CP-GEP stratified 424 stage I/IIA patients (78% of the cohort) according to their risk for recurrence, with five-year relapse-free survival (RFS) rates of 77.8% and 93% for CP-GEP high risk (195 patients) and low risk (229 patients), respectively, and hazard ratio of 3.53 (p-value <0.001). In patients who did not receive SLNB biopsy, CP-GEP captured 6 out of 7 relapses. CONCLUSION: CP-GEP can be used to identify primary cutaneous melanoma patients with a high risk for disease recurrence - especially for stage I/IIA, who are considered low risk by AJCC 8th. These patients may benefit from adjuvant therapy. Also, in the future, when SLNB may become irrelevant, CP-GEP may serve as a risk stratification tool.

High-Dose Ascorbate in Combination with Anti-PD1 Checkpoint Inhibition as Treatment Option for Malignant Melanoma.

Ascorbate acts as a prooxidant when administered parenterally at high supraphysiological doses, which results in the generation of hydrogen peroxide in dependence on oxygen. Most cancer cells are susceptible to the emerging reactive oxygen species (ROS). Accordingly, we evaluated high-dose ascorbate for the treatment of the B16F10 melanoma model. To investigate the effects of ascorbate on the B16F10 cell line in vitro, viability, cellular impedance, and ROS production were analyzed. In vivo, C57BL/6NCrl mice were subcutaneously injected into the right flank with B16F10 cells and tumor-bearing mice were treated intraperitoneally with ascorbate (3 g/kg bodyweight), immunotherapy (anti-programmed cell death protein 1 (PD1) antibody J43; 2 mg/kg bodyweight), or both treatments combined. The efficacy and toxicity were analyzed by measuring the respective tumor sizes and mouse weights accompanied by histological analysis of the protein levels of proliferating cell nuclear antigen (Pcna), glucose transporter 1 (Glut-1), and CD3. Treatment of B16F10 melanoma-carrying mice with high-dose ascorbate yielded plasma levels in the pharmacologically effective range, and ascorbate showed efficacy as a monotherapy and when combined with PD1 inhibition. Our data suggest the applicability of ascorbate as an additional therapeutic agent that can be safely combined with immunotherapy and has the potential to potentiate anti-PD1-based immune checkpoint blockades.

Pulmonary Surfactant Proteins Are Inhibited by Immunoglobulin A Autoantibodies in Severe COVID-19.

Rationale: Coronavirus disease 2019 (COVID-19) can lead to acute respiratory distress syndrome with fatal outcomes. Evidence suggests that dysregulated immune responses, including autoimmunity, are key pathogenic factors. Objectives: To assess whether IgA autoantibodies target lung-specific proteins and contribute to disease severity. Methods: We collected 147 blood, 9 lung tissue, and 36 BAL fluid samples from three tertiary hospitals in Switzerland and one in Germany. Severe COVID-19 was defined by the need to administer oxygen. We investigated the presence of IgA autoantibodies and their effects on pulmonary surfactant in COVID-19 using the following methods: immunofluorescence on tissue samples, immunoprecipitations followed by mass spectrometry on BAL fluid samples, enzyme-linked immunosorbent assays on blood samples, and surface tension measurements with medical surfactant. Measurements and Main Results: IgA autoantibodies targeting pulmonary surfactant proteins B and C were elevated in patients with severe COVID-19 but not in patients with influenza or bacterial pneumonia. Notably, pulmonary surfactant failed to reduce surface tension after incubation with either plasma or purified IgA from patients with severe COVID-19. Conclusions: Our data suggest that patients with severe COVID-19 harbor IgA autoantibodies against pulmonary surfactant proteins B and C and that these autoantibodies block the function of lung surfactant, potentially contributing to alveolar collapse and poor oxygenation.

Vitamin C Deficiency in Blood Samples of COVID-19 Patients.

Coronavirus disease 2019 (COVID-19) is the most notable pandemic of the modern era. A relationship between ascorbate (vitamin C) and COVID-19 severity is well known, whereas the role of other vitamins is less understood. The present study compared the blood levels of four vitamins in a cohort of COVID-19 patients with different severities and uninfected individuals. Serum concentrations of ascorbate, calcidiol, retinol, and α-tocopherol were measured in a cohort of 74 COVID-19 patients and 8 uninfected volunteers. The blood levels were statistically compared and additional co-morbidity factors were considered. COVID-19 patients had significantly lower plasma ascorbate levels than the controls (p-value < 0.001), and further stratification revealed that the controls had higher levels than fatal, critical, and severe COVID-19 cases (p-values < 0.001). However, no such trend was observed for calcidiol, retinol, or α-tocopherol (p-value ≥ 0.093). Survival analysis showed that plasma ascorbate below 11.4 µM was associated with a lengthy hospitalization and a high risk of death. The results indicated that COVID-19 cases had depleted blood ascorbate associated with poor medical conditions, confirming the role of this vitamin in the outcome of COVID-19 infection.

Therapeutic Efficacy of Pharmacological Ascorbate on Braf Inhibitor Resistant Melanoma Cells In Vitro and In Vivo.

High-dose ascorbate paradoxically acts as a pro-oxidant causing the formation of hydrogen peroxide in an oxygen dependent manner. Tumor cells (in particular melanoma cells) show an increased vulnerability to ascorbate induced reactive oxygen species (ROS). Therefore, high-dose ascorbate is a promising pharmacological approach to treating refractory melanomas, e.g., with secondary resistance to targeted BRAF inhibitor therapy. BRAF mutated melanoma cells were treated with ascorbate alone or in combination with the BRAF inhibitor vemurafenib. Viability, cell cycle, ROS production, and the protein levels of phospho-ERK1/2, GLUT-1 and HIF-1α were analyzed. To investigate the treatment in vivo, C57BL/6NCrl mice were subcutaneously injected with D4M.3A (BrafV600E) melanoma cells and treated with intraperitoneal injections of ascorbate with or without vemurafenib. BRAF mutated melanoma cell lines either sensitive or resistant to vemurafenib were susceptible to the induction of cell death by pharmacological ascorbate. Treatment of BrafV600E melanoma bearing mice with ascorbate resulted in plasma levels in the pharmacologically active range and significantly improved the therapeutic effect of vemurafenib. We conclude that intravenous high-dose ascorbate will be beneficial for melanoma patients by interfering with the tumor's energy metabolism and can be safely combined with standard melanoma therapies such as BRAF inhibitors without pharmacological interference.

Early disappearance of tumor antigen-reactive T cells from peripheral blood correlates with superior clinical outcomes in melanoma under anti-PD-1 therapy.

BACKGROUND: Anti-programmed cell death protein 1 (PD-1) antibodies are now routinely administered for metastatic melanoma and for increasing numbers of other cancers, but still only a fraction of patients respond. Better understanding of the modes of action and predictive biomarkers for clinical outcome is urgently required. Cancer rejection is mostly T cell-mediated. We previously showed that the presence of NY-ESO-1-reactive and/or Melan-A-reactive T cells in the blood correlated with prolonged overall survival (OS) of patients with melanoma with a heterogeneous treatment background. Here, we investigated whether such reactive T cells can also be informative for clinical outcomes in metastatic melanoma under PD-1 immune-checkpoint blockade (ICB). METHODS: Peripheral blood T cell stimulation by NY-ESO-1 and Melan-A overlapping peptide libraries was assessed before and during ICB in two independent cohorts of a total of 111 patients with stage IV melanoma. In certain cases, tumor-infiltrating lymphocytes could also be assessed for such responses. These were characterized using intracellular cytokine staining for interferon gamma (IFN-γ), tumor negrosis factor (TNF) and CD107a. Digital pathology analysis was performed to quantify NY-ESO-1 and Melan-A expression by tumors. Endpoints were OS and progression-free survival (PFS). RESULTS: The initial presence in the circulation of NY-ESO-1- or Melan-A-reactive T cells which became no longer detectable during ICB correlated with validated, prolonged PFS (HR:0.1; p>0.0001) and OS (HR:0.2; p=0.021). An evaluation of melanoma tissue from selected cases suggested a correlation between tumor-resident NY-ESO-1- and Melan-A-reactive T cells and disease control, supporting the notion of a therapy-associated sequestration of cells from the periphery to the tumor predominantly in those patients benefitting from ICB. CONCLUSIONS: Our findings suggest a PD-1 blockade-dependent infiltration of melanoma-reactive T cells from the periphery into the tumor and imply that this seminally contributes to effective treatment.

Individualized Proteogenomics Reveals the Mutational Landscape of Melanoma Patients in Response to Immunotherapy.

Immune checkpoint inhibitors are used to restore or augment antitumor immune responses and show great promise in the treatment of melanoma and other types of cancers. However, only a small percentage of patients are fully responsive to immune checkpoint inhibition, mostly due to tumor heterogeneity and primary resistance to therapy. Both of these features are largely driven by the accumulation of patient-specific mutations, pointing to the need for personalized approaches in diagnostics and immunotherapy. Proteogenomics integrates patient-specific genomic and proteomic data to study cancer development, tumor heterogeneity and resistance mechanisms. Using this approach, we characterized the mutational landscape of four clinical melanoma patients. This enabled the quantification of hundreds of sample-specific amino acid variants, among them many that were previously not reported in melanoma. Changes in abundance at the protein and phosphorylation site levels revealed patient-specific over-represented pathways, notably linked to melanoma development (MAPK1 activation) or immunotherapy (NLRP1 inflammasome). Personalized data integration resulted in the prediction of protein drug targets, such as the drugs vandetanib and bosutinib, which were experimentally validated and led to a reduction in the viability of tumor cells. Our study emphasizes the potential of proteogenomic approaches to study personalized mutational landscapes, signaling networks and therapy options.

Comparative Analysis of the Antitumor Activity of Cis- and Trans-Resveratrol in Human Cancer Cells with Different p53 Status.

Resveratrol, a natural plant phytoalexin, is produced in response to fungal infection or- UV irradiation. It exists as an isomeric pair with cis- and trans-conformation. Whereas multiple physiological effects of the trans-form, including a pronounced anti-tumoral activity, are nowadays elucidated, much less knowledge exists concerning the cis-isomer. In our work, we analyzed the antiproliferative and cytotoxic properties of cis-resveratrol in four different human tumor entities in direct comparison to trans-resveratrol. We used human cell lines as tumor models for hepatocellular carcinoma (HCC; HepG2, Hep3B), colon carcinoma (HCT-116, HCT-116/p53(-/-)), pancreatic carcinoma (Capan-2, MiaPaCa-2), and renal cell carcinoma (A498, SN12C). Increased cytotoxicity in all investigated tumor cells was observed for the trans-isomer. To verify possible effects of the tumor suppressor p53 on resveratrol-induced cell death, we used wild type and p53-deleted or -mutated cell lines for every tested tumor entity. Applying viability and cytotoxicity assays, we demonstrated a differential, dose-dependent sensitivity towards cis- or trans-resveratrol among the respective tumor types.

Ex Vivo Evaluation of the Sepsis Triple Therapy High-Dose Vitamin C in Combination with Vitamin B1 and Hydrocortisone in a Human Peripheral Blood Mononuclear Cells (PBMCs) Model.

Sepsis is an extremely complex clinical syndrome, usually involving an excessive inflammatory response including an overshooting cytokine release that damages tissue and organs of the patient. Due to the severity of this condition, it is estimated that over 11 million people die from sepsis each year. Despite intensive research in the field, there is still no specific therapy for sepsis. Many sepsis patients show a marked deficiency of vitamin C. 9 out of 10 sepsis patients have a hypovitaminosis C, and every third patient even shows a clinical deficiency in the scurvy range. In addition, low vitamin C levels of intensive care sepsis patients correlate with a higher need for vasopressors, higher Sequential Organ Failure Assessment (SOFA) scores, and increased mortality. Based on this observation and the conducted clinical trials using vitamin C as sepsis therapy in intensive care patients, the aim of the present ex vivo study was to evaluate the effects of high-dose vitamin C alone and in a triple combination supplemented with vitamin B1 (thiamine) and hydrocortisone on the lipopolysaccharide (LPS)-induced cytokine response in peripheral blood mononuclear cells (PBMCs) from healthy human donors. We found that all corticosteroid combinations strongly reduced the cytokine response on RNA- and protein levels, while high-dose vitamin C alone significantly diminished the PBMC mediated secretion of the cytokines interleukin (IL)-10, IL-23, and monocyte chemo-attractant protein (MCP-1), which mediate the inflammatory response. However, vitamin C showed no enhancing effect on the secretion of further cytokines studied. This data provides important insights into the possible immunomodulatory function of vitamin C in an ex vivo setting of human PBMCs and the modulation of their cytokine profile in the context of sepsis. Since vitamin C is a vital micronutrient, the restoration of physiologically adequate concentrations should be integrated into routine sepsis therapy, and the therapeutic effects of supraphysiological concentrations of vitamin C in sepsis patients should be further investigated in clinical trials.