Functional Test of a Naturally Occurred Tumor Modifier Gene Provides Insights to Melanoma Development.

Occurrence of degenerative interactions is thought to serve as a mechanism underlying hybrid unfitness. However, the molecular mechanisms underpinning the genetic interaction and how they contribute to overall hybrid incompatibilities are limited to only a handful of examples. A vertebrate model organism, Xiphophorus , is used to study hybrid dysfunction and it has been shown from this model that diseases, such as melanoma, can occur in certain interspecies hybrids. Melanoma development is due to hybrid inheritance of an oncogene, xmrk , and loss of a co-evolved tumor modifier. It was recently found that adgre5 , a G protein-coupled receptor involved in cell adhesion, is a tumor regulator gene in naturally hybridizing Xiphophorus species X. birchmanni and X. malinche . We hypothesized that one of the two parental alleles of adgre5 is involved in regulation of cell proliferation, migration and melanomagenesis. Accordingly, we assessed the function of adgre5 alleles from each parental species of the melanoma-bearing hybrids using in vitro cell proliferation and migration assays. In addition, we expressed each adgre5 allele with the xmrk oncogene in transgenic medaka. We found that cells transfected with the X. birchmanni adgre5 exhibited decreased proliferation and migration compared to those with the X. malinche allele. Moreover, X. birchmanni allele of adgre5 completely inhibited melanoma development in xmrk transgenic medaka, while X. malinche adgre5 expression did not exhibit melanoma suppressive activity in medaka. These findings showed that adgre5 is a natural melanoma suppressor and provide new insight in melanoma etiology.

Loss of p14 diminishes immunogenicity in melanoma via non-canonical Wnt signaling by reducing the peptide surface density.

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 p14ARF and p16INK4a, belongs to the most frequently inactivated gene loci in melanoma and leads to decreased T cell infiltration. While the role of p16INK4a has been extensively investigated, knowledge about p14ARF in melanoma is scarce. In this study, we elucidate the impact of reduced p14ARF expression on melanoma immunogenicity. Knockdown of p14ARF 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 p14ARF 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.

7-Dehydrocholesterol is an endogenous suppressor of ferroptosis.

Ferroptosis is a form of cell death that has received considerable attention not only as a means to eradicate defined tumour entities but also because it provides unforeseen insights into the metabolic adaptation that tumours exploit to counteract phospholipid oxidation1,2. Here, we identify proferroptotic activity of 7-dehydrocholesterol reductase (DHCR7) and an unexpected prosurvival function of its substrate, 7-dehydrocholesterol (7-DHC). Although previous studies suggested that high concentrations of 7-DHC are cytotoxic to developing neurons by favouring lipid peroxidation3, we now show that 7-DHC accumulation confers a robust prosurvival function in cancer cells. Because of its far superior reactivity towards peroxyl radicals, 7-DHC effectively shields (phospho)lipids from autoxidation and subsequent fragmentation. We provide validation in neuroblastoma and Burkitt's lymphoma xenografts where we demonstrate that the accumulation of 7-DHC is capable of inducing a shift towards a ferroptosis-resistant state in these tumours ultimately resulting in a more aggressive phenotype. Conclusively, our findings provide compelling evidence of a yet-unrecognized antiferroptotic activity of 7-DHC as a cell-intrinsic mechanism that could be exploited by cancer cells to escape ferroptosis.

Thiol starvation triggers melanoma state switching in an ATF4 and NRF2-dependent manner.

The cystine/glutamate antiporter xCT is an important source of cysteine for cancer cells. Once taken up, cystine is reduced to cysteine and serves as a building block for the synthesis of glutathione, which efficiently protects cells from oxidative damage and prevents ferroptosis. As melanomas are particularly exposed to several sources of oxidative stress, we investigated the biological role of cysteine and glutathione supply by xCT in melanoma. xCT activity was abolished by genetic depletion in the Tyr::CreER; BrafCA; Ptenlox/+ melanoma model and by acute cystine withdrawal in melanoma cell lines. Both interventions profoundly impacted melanoma glutathione levels, but they were surprisingly well tolerated by murine melanomas in vivo and by most human melanoma cell lines in vitro. RNA sequencing of human melanoma cells revealed a strong adaptive upregulation of NRF2 and ATF4 pathways, which orchestrated the compensatory upregulation of genes involved in antioxidant defence and de novo cysteine biosynthesis. In addition, the joint activation of ATF4 and NRF2 triggered a phenotypic switch characterized by a reduction of differentiation genes and induction of pro-invasive features, which was also observed after erastin treatment or the inhibition of glutathione synthesis. NRF2 alone was capable of inducing the phenotypic switch in a transient manner. Together, our data show that cystine or glutathione levels regulate the phenotypic plasticity of melanoma cells by elevating ATF4 and NRF2.

LRP8-mediated selenocysteine uptake is a targetable vulnerability in MYCN-amplified neuroblastoma.

Ferroptosis has emerged as an attractive strategy in cancer therapy. Understanding the operational networks regulating ferroptosis may unravel vulnerabilities that could be harnessed for therapeutic benefit. Using CRISPR-activation screens in ferroptosis hypersensitive cells, we identify the selenoprotein P (SELENOP) receptor, LRP8, as a key determinant protecting MYCN-amplified neuroblastoma cells from ferroptosis. Genetic deletion of LRP8 leads to ferroptosis as a result of an insufficient supply of selenocysteine, which is required for the translation of the antiferroptotic selenoprotein GPX4. This dependency is caused by low expression of alternative selenium uptake pathways such as system Xc- . The identification of LRP8 as a specific vulnerability of MYCN-amplified neuroblastoma cells was confirmed in constitutive and inducible LRP8 knockout orthotopic xenografts. These findings disclose a yet-unaccounted mechanism of selective ferroptosis induction that might be explored as a therapeutic strategy for high-risk neuroblastoma and potentially other MYCN-amplified entities.

The integrated stress response effector ATF4 is an obligatory metabolic activator of NRF2.

The redox regulator NRF2 becomes activated upon oxidative and electrophilic stress and orchestrates a response program associated with redox regulation, metabolism, tumor therapy resistance, and immune suppression. Here, we describe an unrecognized link between the integrated stress response (ISR) and NRF2 mediated by the ISR effector ATF4. The ISR is commonly activated after starvation or ER stress and plays a central role in tissue homeostasis and cancer plasticity. ATF4 increases NRF2 transcription and induces the glutathione-degrading enzyme CHAC1, which we now show to be critically important for maintaining NRF2 activation. In-depth analyses reveal that NRF2 supports ATF4-induced cells by increasing cystine uptake via the glutamate-cystine antiporter xCT. In addition, NRF2 upregulates genes mediating thioredoxin usage and regeneration, thus balancing the glutathione decrease. In conclusion, we demonstrate that the NRF2 response serves as second layer of the ISR, an observation highly relevant for the understanding of cellular resilience in health and disease.

Multimodal treatment and immune checkpoint inhibition in sinonasal mucosal melanoma: real-world data of a retrospective, single-center study.

PURPOSE: Local failure and distant metastases occur frequently in sinonasal mucosal melanoma (SNMM). Response rates to chemotherapy are low and targetable mutations are rarely detected. However, there is increasing data indicating efficacy of immune checkpoint inhibition (ICI). The aim of this retrospective monocenter study was to assess the mutational landscape and to evaluate the outcome of surgical treatment and ICI in SNMM in a real-world setting. METHODS: Thirty-eight SNMM patients being treated between 1999 and 2020 at our institution were retrospectively reviewed. Survival curves were generated according to Kaplan-Meier and compared by the log-rank test. RESULTS: Local failure was seen in 60% of patients treated in a curative intent. Overall, 24% of all patients suffered from regional and 66% from distant metastases. Next generation sequencing revealed mutations of BRAF, NRAS and KRAS. One out of three patients treated with a primary ICI showed a complete response (CR) and two showed progressive disease. Eleven patients received ICI as a palliative treatment. CR could be observed in three patients and stable disease in one patient. In the whole study population, the 5-year overall survival rate (OS) was 26%. OS was better for patients who received ICI during the course of disease. CONCLUSIONS: Recurrences and distant metastases are frequent in SNMM. Durable CR could be observed after primary and palliative ICI. Therefore, ICI in a palliative, adjuvant or even neoadjuvant setting might play a promising role in SNMM therapy while targetable mutations are rarely detected.

Predicting Microenvironment in CXCR4- and FAP-Positive Solid Tumors-A Pan-Cancer Machine Learning Workflow for Theranostic Target Structures.

(1) Background: C-X-C Motif Chemokine Receptor 4 (CXCR4) and Fibroblast Activation Protein Alpha (FAP) are promising theranostic targets. However, it is unclear whether CXCR4 and FAP positivity mark distinct microenvironments, especially in solid tumors. (2) Methods: Using Random Forest (RF) analysis, we searched for entity-independent mRNA and microRNA signatures related to CXCR4 and FAP overexpression in our pan-cancer cohort from The Cancer Genome Atlas (TCGA) database-representing n = 9242 specimens from 29 tumor entities. CXCR4- and FAP-positive samples were assessed via StringDB cluster analysis, EnrichR, Metascape, and Gene Set Enrichment Analysis (GSEA). Findings were validated via correlation analyses in n = 1541 tumor samples. TIMER2.0 analyzed the association of CXCR4 / FAP expression and infiltration levels of immune-related cells. (3) Results: We identified entity-independent CXCR4 and FAP gene signatures representative for the majority of solid cancers. While CXCR4 positivity marked an immune-related microenvironment, FAP overexpression highlighted an angiogenesis-associated niche. TIMER2.0 analysis confirmed characteristic infiltration levels of CD8+ cells for CXCR4-positive tumors and endothelial cells for FAP-positive tumors. (4) Conclusions: CXCR4- and FAP-directed PET imaging could provide a non-invasive decision aid for entity-agnostic treatment of microenvironment in solid malignancies. Moreover, this machine learning workflow can easily be transferred towards other theranostic targets.

Extending the Mass Spectrometry-Detectable Landscape of MHC Peptides by Use of Restricted Access Material.

Mass spectrometry-based immunopeptidomics enables the comprehensive identification of major histocompatibility complex (MHC) peptides from a cell culture as well as from tissue or tumor samples and is applied for the identification of tumor-specific and viral T-cell epitopes. Although mass spectrometry is generally considered an "unbiased" method for MHC peptide identification, the physicochemical properties of MHC peptides can greatly influence their detectability. Here, we demonstrate that highly hydrophobic peptides are lost during sample preparation when C18 solid-phase extraction (SPE) is used for separating MHC peptides from proteins. To overcome this limitation, we established an optimized protocol involving restricted access material (RAM). Compared to C18-SPE, RAM-SPE improved the overall MHC peptide recovery and extended the landscape of mass spectrometry-detectable MHC peptides toward more hydrophobic peptides.

Effect of stress-induced polyploidy on melanoma reprogramming and therapy resistance.

Melanomas and their precursors, the melanocytes, are frequently exposed to UV due to their anatomic location, leading to DNA damage and reactive oxygen stress related harm. Such damage can result in multinucleation or polyploidy, in particularly in presence of mitotic or cell division failure. As a consequence, the cell encounters either of two fates: mitotic catastrophe, resulting in cell death, or survival and recovery, the latter occurring less frequently. However, when cells manage to recover in an polyploid state, they have often acquired new features, which allow them to tolerate and adapt to oncogene- or therapy induced stress. This review focuses on polyploidy inducers in melanoma and their effects on transcriptional reprogramming and phenotypic adaptation as well as the relevance of polyploid melanoma cells for therapy resistance.

Subgroup-Independent Mapping of Renal Cell Carcinoma-Machine Learning Reveals Prognostic Mitochondrial Gene Signature Beyond Histopathologic Boundaries.

Background: Renal cell carcinoma (RCC) is divided into three major histopathologic groups-clear cell (ccRCC), papillary (pRCC) and chromophobe RCC (chRCC). We performed a comprehensive re-analysis of publicly available RCC datasets from the TCGA (The Cancer Genome Atlas) database, thereby combining samples from all three subgroups, for an exploratory transcriptome profiling of RCC subgroups. Materials and Methods: We used FPKM (fragments per kilobase per million) files derived from the ccRCC, pRCC and chRCC cohorts of the TCGA database, representing transcriptomic data of 891 patients. Using principal component analysis, we visualized datasets as t-SNE plot for cluster detection. Clusters were characterized by machine learning, resulting gene signatures were validated by correlation analyses in the TCGA dataset and three external datasets (ICGC RECA-EU, CPTAC-3-Kidney, and GSE157256). Results: Many RCC samples co-clustered according to histopathology. However, a substantial number of samples clustered independently from histopathologic origin (mixed subgroup)-demonstrating divergence between histopathology and transcriptomic data. Further analyses of mixed subgroup via machine learning revealed a predominant mitochondrial gene signature-a trait previously known for chRCC-across all histopathologic subgroups. Additionally, ccRCC samples from mixed subgroup presented an inverse correlation of mitochondrial and angiogenesis-related genes in the TCGA and in three external validation cohorts. Moreover, mixed subgroup affiliation was associated with a highly significant shorter overall survival for patients with ccRCC-and a highly significant longer overall survival for chRCC patients. Conclusions: Pan-RCC clustering according to RNA-sequencing data revealed a distinct histology-independent subgroup characterized by strengthened mitochondrial and weakened angiogenesis-related gene signatures. Moreover, affiliation to mixed subgroup went along with a significantly shorter overall survival for ccRCC and a longer overall survival for chRCC patients. Further research could offer a therapy stratification by specifically addressing the mitochondrial metabolism of such tumors and its microenvironment.

NRF2 Enables EGFR Signaling in Melanoma Cells.

Receptor tyrosine kinases (RTK) are rarely mutated in cutaneous melanoma, but the expression and activation of several RTK family members are associated with a proinvasive phenotype and therapy resistance. Epidermal growth factor receptor (EGFR) is a member of the RTK family and is only expressed in a subgroup of melanomas with poor prognosis. The insight into regulators of EGFR expression and activation is important for the understanding of the development of this malignant melanoma phenotype. Here, we describe that the transcription factor NRF2, the master regulator of the oxidative and electrophilic stress response, mediates the expression and activation of EGFR in melanoma by elevating the levels of EGFR as well as its ligands EGF and TGFα. ChIP sequencing data show that NRF2 directly binds to the promoter of EGF, which contains a canonical antioxidant response element. Accordingly, EGF is induced by oxidative stress and is also increased in lung adenocarcinoma and head and neck carcinoma with mutationally activated NRF2. In contrast, regulation of EGFR and TGFA occurs by an indirect mechanism, which is enabled by the ability of NRF2 to block the activity of the melanocytic lineage factor MITF in melanoma. MITF effectively suppresses EGFR and TGFA expression and therefore serves as link between NRF2 and EGFR. As EGFR was previously described to stimulate NRF2 activity, the mutual activation of NRF2 and EGFR pathways was investigated. The presence of NRF2 was necessary for full EGFR pathway activation, as NRF2-knockout cells showed reduced AKT activation in response to EGF stimulation compared to controls. Conversely, EGF led to the nuclear localization and activation of NRF2, thereby demonstrating that NRF2 and EGFR are connected in a positive feedback loop in melanoma. In summary, our data show that the EGFR-positive melanoma phenotype is strongly supported by NRF2, thus revealing a novel maintenance mechanism for this clinically challenging melanoma subpopulation.

NRF2-dependent stress defense in tumor antioxidant control and immune evasion.

The transcription factor NRF2 is known as the master regulator of the oxidative stress response. Tumor entities presenting oncogenic activation of NRF2, such as lung adenocarcinoma, are associated with drug resistance, and accumulating evidence demonstrates its involvement in immune evasion. In other cancer types, the KEAP1/NRF2 pathway is not commonly mutated, but NRF2 is activated by other means such as radiation, oncogenic activity, cytokines, or other pro-oxidant triggers characteristic of the tumor niche. The obvious effect of stress-activated NRF2 is the protection from oxidative or electrophilic damage and the adaptation of the tumor metabolism to changing conditions. However, data from melanoma also reveal a role of NRF2 in modulating differentiation and suppressing anti-tumor immunity. This review summarizes the function of NRF2 in this tumor entity and discusses the implications for current tumor therapies.

Emerging aspects in the regulation of ferroptosis.

Lipid peroxidation has been associated with a wide array of (patho)physiological conditions. Remarkably, in the last few years, a novel cell death modality termed ferroptosis was recognized as a process initiated by iron-dependent oxidation of lipids. The sensitivity to ferroptosis is determined by the activity of antioxidant systems working on the repair of oxidized phospholipids and also metabolic pathways controlling the availability of substrates susceptible to lipid peroxidation. Non-enzymatic antioxidants such as vitamin E, which has long been acknowledged as an efficient inhibitor of lipid peroxidation, play an important and often neglected role in subverting ferroptosis. Recent works dissecting the mechanisms that determine ferroptosis sensitivity have provided further insights into the contribution of alternative metabolic pathways able to suppress lipid peroxidation. Specifically, the role of ubiquinone and tetrahydrobiopterin (BH4) has been brought forth, with the identification of specific enzymatic systems responsible for their regeneration, as critical factors suppressing ferroptosis. Therefore, in the present manuscript, we address these emerging concepts and propose that the characterization of these antioxidant repair mechanisms will not only open a new understanding of disease conditions where ferroptosis plays a role but also offer opportunities to identify and sensitize cells to ferroptosis in the context of cancer treatment.

The transcription factor NRF2 enhances melanoma malignancy by blocking differentiation and inducing COX2 expression.

The transcription factor NRF2 is the major mediator of oxidative stress responses and is closely connected to therapy resistance in tumors harboring activating mutations in the NRF2 pathway. In melanoma, such mutations are rare, and it is unclear to what extent melanomas rely on NRF2. Here we show that NRF2 suppresses the activity of the melanocyte lineage marker MITF in melanoma, thereby reducing the expression of pigmentation markers. Intriguingly, we furthermore identified NRF2 as key regulator of immune-modulating genes, linking oxidative stress with the induction of cyclooxygenase 2 (COX2) in an ATF4-dependent manner. COX2 is critical for the secretion of prostaglandin E2 and was strongly induced by H2O2 or TNFα only in presence of NRF2. Induction of MITF and depletion of COX2 and PGE2 were also observed in NRF2-deleted melanoma cells in vivo. Furthermore, genes corresponding to the innate immune response such as RSAD2 and IFIH1 were strongly elevated in absence of NRF2 and coincided with immune evasion parameters in human melanoma datasets. Even in vitro, NRF2 activation or prostaglandin E2 supplementation blunted the induction of the innate immune response in melanoma cells. Transcriptome analyses from lung adenocarcinomas indicate that the observed link between NRF2 and the innate immune response is not restricted to melanoma.

IFN-gamma-induced PD-L1 expression in melanoma depends on p53 expression.

BACKGROUND: Immune checkpoint inhibition and in particular anti-PD-1 immunotherapy have revolutionized the treatment of advanced melanoma. In this regard, higher tumoral PD-L1 protein (gene name: CD274) expression is associated with better clinical response and increased survival to anti-PD-1 therapy. Moreover, there is increasing evidence that tumor suppressor proteins are involved in immune regulation and are capable of modulating the expression of immune checkpoint proteins. Here, we determined the role of p53 protein (gene name: TP53) in the regulation of PD-L1 expression in melanoma. METHODS: We analyzed publicly available mRNA and protein expression data from the cancer genome/proteome atlas and performed immunohistochemistry on tumors with known TP53 status. Constitutive and IFN-É£-induced PD-L1 expression upon p53 knockdown in wildtype, TP53-mutated or JAK2-overexpressing melanoma cells or in cells, in which p53 was rendered transcriptionally inactive by CRISPR/Cas9, was determined by immunoblot or flow cytometry. Similarly, PD-L1 expression was investigated after overexpression of a transcriptionally-impaired p53 (L22Q, W23S) in TP53-wt or a TP53-knockout melanoma cell line. Immunoblot was applied to analyze the IFN-É£ signaling pathway. RESULTS: For TP53-mutated tumors, an increased CD274 mRNA expression and a higher frequency of PD-L1 positivity was observed. Interestingly, positive correlations of IFNG mRNA and PD-L1 protein in both TP53-wt and -mutated samples and of p53 and PD-L1 protein suggest a non-transcriptional mode of action of p53. Indeed, cell line experiments revealed a diminished IFN-É£-induced PD-L1 expression upon p53 knockdown in both wildtype and TP53-mutated melanoma cells, which was not the case when p53 wildtype protein was rendered transcriptionally inactive or by ectopic expression of p53L22Q,W23S, a transcriptionally-impaired variant, in TP53-wt cells. Accordingly, expression of p53L22Q,W23S in a TP53-knockout melanoma cell line boosted IFN-É£-induced PD-L1 expression. The impaired PD-L1-inducibility after p53 knockdown was associated with a reduced JAK2 expression in the cells and was almost abrogated by JAK2 overexpression. CONCLUSIONS: While having only a small impact on basal PD-L1 expression, both wildtype and mutated p53 play an important positive role for IFN-É£-induced PD-L1 expression in melanoma cells by supporting JAK2 expression. Future studies should address, whether p53 expression levels might influence response to anti-PD-1 immunotherapy.

Role of melanoma inhibitory activity in melanocyte senescence.

The protein melanoma inhibitory activity (MIA) is known to be expressed in melanoma and to support melanoma progression. Interestingly, previous studies also observed the expression of MIA in nevi. Concentrating on these findings, we revealed that MIA expression is correlated with a senescent state in melanocytes. Induction of replicative or oncogene-induced senescence resulted in increased MIA expression in vitro. Notably, MIA knockdown in senescent melanocytes reduced the percentage of senescence-associated beta-Gal-positive cells and enhanced proliferation. Using the melanoma mouse model Tg(Grm1), MIA-deficient mice supported the impact of MIA on senescence by showing a significantly earlier tumor onset compared to controls. In melanocytes, MIA knockdown led to a downregulation of the cell cycle inhibitor p21 in vitro and in vivo. In contrast, after induction of hTERT in human melanoma cells, p21 regulation by MIA was lost. In summary, our data show for the first time that MIA is a regulator of cellular senescence in human and murine melanocytes.