In Silico Transcriptomic Expression of MSR1 in Solid Tumors Is Associated with Responses to Anti-PD1 and Anti-CTLA4 Therapies.

Immuno-oncology has gained momentum with the approval of antibodies with clinical activities in different indications. Unfortunately, for anti-PD (L)1 agents in monotherapy, only half of the treated population achieves a clinical response. For other agents, such as anti-CTLA4 antibodies, no biomarkers exist, and tolerability can limit administration. In this study, using publicly available genomic datasets, we evaluated the expression of the macrophage scavenger receptor-A (SR-A) (MSR1) and its association with a response to check-point inhibitors (CPI). MSR1 was associated with the presence of macrophages, dendritic cells (DCs) and neutrophils in most of the studied indications. The presence of MSR1 was associated with macrophages with a pro-tumoral phenotype and correlated with TIM3 expression. MSR1 predicted favorable overall survival in patients treated with anti-PD1 (HR: 0.56, FDR: 1%, p = 2.6 × 10-5), anti PD-L1 (HR: 0.66, FDR: 20%, p = 0.00098) and anti-CTLA4 (HR: 0.37, FDR: 1%, p = 4.8 × 10-5). When specifically studying skin cutaneous melanoma (SKCM), we observed similar effects for anti-PD1 (HR: 0.65, FDR: 50%, p = 0.0072) and anti-CTLA4 (HR: 0.35, FDR: 1%, p = 4.1 × 10-5). In a different dataset of SKCM patients, the expression of MSR1 predicted a clinical response to anti-CTLA4 (AUC: 0.61, p = 2.9 × 10-2). Here, we describe the expression of MSR1 in some solid tumors and its association with innate cells and M2 phenotype macrophages. Of note, the presence of MSR1 predicted a response to CPI and, particularly, anti-CTLA4 therapies in different cohorts of patients. Future studies should prospectively explore the association of MSR1 expression and the response to anti-CTLA4 strategies in solid tumors.

Deciphering cutaneous melanoma prognosis through LDL metabolism: Single-cell transcriptomics analysis via 101 machine learning algorithms.

Cutaneous melanoma poses a formidable challenge within the field of oncology, marked by its aggressive nature and capacity for metastasis. Despite extensive research uncovering numerous genetic and molecular contributors to cutaneous melanoma development, there remains a critical knowledge gap concerning the role of lipids, notably low-density lipoprotein (LDL), in this lethal skin cancer. This article endeavours to bridge this knowledge gap by delving into the intricate interplay between LDL metabolism and cutaneous melanoma, shedding light on how lipids influence tumour progression, immune responses and potential therapeutic avenues. Genes associated with LDL metabolism were extracted from the GSEA database. We acquired and analysed single-cell sequencing data (GSE215120) and bulk-RNA sequencing data, including the TCGA data set, GSE19234, GSE22153 and GSE65904. Our analysis unveiled the heterogeneity of LDL across various cell types at the single-cell sequencing level. Additionally, we constructed an LDL-related signature (LRS) using machine learning algorithms, incorporating differentially expressed genes and highly correlated genes. The LRS serves as a valuable tool for assessing the prognosis, immunity and mutation status of patients with cutaneous melanoma. Furthermore, we conducted experiments on A375 and WM-115 cells to validate the function of PPP2R1A, a pivotal gene within the LRS. Our comprehensive approach, combining advanced bioinformatics analyses with an extensive review of current literature, presents compelling evidence regarding the significance of LDL within the cutaneous melanoma microenvironment.

White horses - non-coding sequences drive premature hair greying and predisposition to melanoma.

The Grey allele in horses is causing premature hair greying and susceptibility to melanoma. The causal mutation is a 4.6 kb tandem duplication in intron 6 of the Syntaxin 17 gene. A recent study demonstrated that the most common allele at the Grey locus (G3) involves three tandem copies of this sequence, whilst a more rare allele (G2) has two tandem copies and the wild-type allele (G1) only one copy. The G3 allele is causing fast greying and high incidence of skin melanoma, whereas the G2 allele is causing slow greying and no obvious increase in melanoma incidence. Further somatic copy number expansion has been documented in melanoma tissue from Grey horses. Functional studies showed that this intronic sequence acts as a weak melanocyte-specific enhancer that becomes substantially stronger by the copy number expansion. The Grey mutation is associated with upregulated expression of both Syntaxin 17 and the neighbouring NR4A3 gene in Grey horse melanomas. It is still an open question which of these genes is most important for the phenotypic effects or if causality is due to the combined effect of upregulation of both genes. Interestingly, RNAseq data in the Human Protein Atlas give support for a possible role of NR4A3 because it is particularly upregulated in human skin cancer, and it belongs to a cluster of genes associated with skin cancer and melanin biosynthesis. The Grey mutation and its association with melanoma provide a possibility to study the path to tumour development in numerous Grey horses carrying exactly the same predisposing mutation.

Deciphering the role of wound healing genes in skin cutaneous melanoma: Insights into expression, methylation, mutations, and therapeutic implications.

Skin Cutaneous Melanoma (SKCM) is a form of cancer that originates in the pigment-producing cells, known as melanocytes, of the skin. Delay wound healing is often correlated with the occurrence of and progression of SKCM. In this comprehensive study, we investigated the intricate roles of two important wound healing genes in SKCM, including Matrix Metalloproteinase-2 (MMP2) and Matrix Metalloproteinase-9 (MMP9). Through a multi-faceted approach, we collected clinical samples, conducted molecular experiments, including RT-qPCR, bisulphite sequencing, cell culture, cell Counting Kit-8, colony formation, and wound healing assays. Beside this, we also used various other databases/tools/approaches for additional analysis including, UALCAN, GEPIA, HPA, MEXPRESS, cBioPortal, KM plotter, DrugBank, and molecular docking. Our results revealed a significant up-regulation of MMP2 and MMP9 in SKCM tissues compared to normal counterparts. Moreover, promoter methylation analysis suggested an epigenetic regulatory mechanism. Validations using TCGA datasets and immunohistochemistry emphasized the clinical relevance of MMP2 and MMP9 dysregulation. Functional assays demonstrated their synergistic impact on proliferation and migration in SKCM cells. Furthermore, we identified potential therapeutic candidates, Estradiol and Calcitriol, through drug prediction and molecular docking analyses. These compounds exhibited binding affinities, suggesting their potential as MMP2/MMP9 inhibitors. Overall, our study elucidates the diagnostic, prognostic, and therapeutic implications of MMP2 and MMP9 in SKCM, shedding light on their complex interplay in SKCM occurrence and progression.

Molecular patterns of resistance to immune checkpoint blockade in melanoma.

Immune checkpoint blockade (ICB) has improved outcome for patients with metastatic melanoma but not all benefit from treatment. Several immune- and tumor intrinsic features are associated with clinical response at baseline. However, we need to further understand the molecular changes occurring during development of ICB resistance. Here, we collect biopsies from a cohort of 44 patients with melanoma after progression on anti-CTLA4 or anti-PD1 monotherapy. Genetic alterations of antigen presentation and interferon gamma signaling pathways are observed in approximately 25% of ICB resistant cases. Anti-CTLA4 resistant lesions have a sustained immune response, including immune-regulatory features, as suggested by multiplex spatial and T cell receptor (TCR) clonality analyses. One anti-PD1 resistant lesion harbors a distinct immune cell niche, however, anti-PD1 resistant tumors are generally immune poor with non-expanded TCR clones. Such immune poor microenvironments are associated with melanoma cells having a de-differentiated phenotype lacking expression of MHC-I molecules. In addition, anti-PD1 resistant tumors have reduced fractions of PD1+ CD8+ T cells as compared to ICB naïve metastases. Collectively, these data show the complexity of ICB resistance and highlight differences between anti-CTLA4 and anti-PD1 resistance that may underlie differential clinical outcomes of therapy sequence and combination.

Combined therapy of dabrafenib and an anti-HER2 antibody-drug conjugate for advanced BRAF-mutant melanoma.

BACKGROUND: Melanoma is the most lethal skin cancer characterized by its high metastatic potential. In the past decade, targeted and immunotherapy have brought revolutionary survival benefits to patients with advanced and metastatic melanoma, but these treatment responses are also heterogeneous and/or do not achieve durable responses. Therefore, novel therapeutic strategies for improving outcomes remain an unmet clinical need. The aim of this study was to evaluate the therapeutic potential and underlying molecular mechanisms of RC48, a novel HER2-target antibody drug conjugate, either alone or in combination with dabrafenib, a V600-mutant BRAF inhibitor, for the treatment of advanced BRAF-mutant cutaneous melanoma. METHODS: We evaluated the therapeutic efficacy of RC48, alone or in combination with dabrafenib, in BRAF-mutant cutaneous melanoma cell lines and cell-derived xenograft (CDX) models. We also conducted signaling pathways analysis and global mRNA sequencing to explore mechanisms underlying the synergistic effect of the combination therapy. RESULTS: Our results revealed the expression of membrane-localized HER2 in melanoma cells. RC48 effectively targeted and inhibited the growth of HER2-positive human melanoma cell lines and corresponding CDX models. When used RC48 and dabrafenib synergically induced tumor regression together in human BRAF-mutant melanoma cell lines and CDX models. Mechanically, our results demonstrated that the combination therapy induced apoptosis and cell cycle arrest while suppressing cell motility in vitro. Furthermore, global RNA sequencing analysis demonstrated that the combination treatment led to the downregulation of several key signaling pathways, including the PI3K-AKT pathway, MAPK pathway, AMPK pathway, and FOXO pathway. CONCLUSION: These findings establish a preclinical foundation for the combined use of an anti-HER2 drug conjugate and a BRAF inhibitor in the treatment of BRAF-mutant cutaneous melanoma.

KLF4 suppresses anticancer effects of brusatol via transcriptional upregulating NCK2 expression in melanoma.

Brusatol (Bru), a main extract from traditional Chinese medicine Brucea javanica, has been reported to exist antitumor effect in many tumors including melanoma. However, the underlying mechanism in its anti-melanoma effect still need further exploration. Here, we reported that the protein expression of KLF4 in melanoma cells were significantly downregulated in response to brusatol treatment. Overexpression of KLF4 suppressed brusatol-induced melanoma cell apoptosis; while knockdown of KLF4 enhanced antitumor effects of brusatol on melanoma cells not only in vitro but also in vivo. Further studies on the mechanism revealed that KLF4 bound to the promoter of NCK2 directly and facilitated NCK2 transcription, which suppressed the antitumor effect of brusatol on melanoma. Furthermore, our findings showed that miR-150-3p was dramatically upregulated under brusatol treatment which resulted in the downregulation of KLF4. Our results suggested that the miR-150-3p/KLF4/NCK2 axis might play an important role in the antitumour effects of brusatol in melanoma.

Strategies for improving the performance of prediction models for response to immune checkpoint blockade therapy in cancer.

Immune checkpoint blockade (ICB) therapy holds promise for bringing long-lasting clinical gains for the treatment of cancer. However, studies show that only a fraction of patients respond to the treatment. In this regard, it is valuable to develop gene expression signatures based on RNA sequencing (RNAseq) data and machine learning methods to predict a patient's response to the ICB therapy, which contributes to more personalized treatment strategy and better management of cancer patients. However, due to the limited sample size of ICB trials with RNAseq data available and the vast number of candidate gene expression features, it is challenging to develop well-performed gene expression signatures. In this study, we used several published melanoma datasets and investigated approaches that can improve the construction of gene expression-based prediction models. We found that merging datasets from multiple studies and incorporating prior biological knowledge yielded prediction models with higher predictive accuracies. Our finding suggests that these two strategies are of high value to identify ICB response biomarkers in future studies.

Artesunate attenuates the tumorigenesis of choroidal melanoma via inhibiting EFNA3 through Stat3/Akt signaling pathway.

PURPOSE: Choroidal melanoma (CM), a kind of malignant tumor, is the main type of Uveal melanoma and one half of CM patients develop metastases. As a member of Eph/ephrin pathway that plays vital role in tumors, EphrinA3 (EFNA3) has been proved to promote tumorigenesis in many tumors. But the effect of EFNA3 in CM has not been studied yet. Through inhibiting angiogenesis, inducing apoptosis and autophagy and so on, Artesunate (ART) plays a key anti-tumor role in many tumors, including CM. However, the exact mechanisms of anti-tumor in CM remain unclear. METHODS: The UALCAN and TIMER v2.0 database analyzed the role of EFNA3 in CM patients. Quantitative real time polymerase chain reaction (qPCR) and Western blot were used to detect the expression of EFNA3 in CM. The growth ability of CM was tested by clonogenic assay and Cell counting kit-8 assay, and the migration ability using Transwell assay. RESULTS: Our results found EFNA3 boosted CM cells' growth and migration through activating Stat3/Akt signaling pathway, while ART inhibited the tumor promoting effect of CM via downregulating EFNA3. In xenograft tumor model, EFNA3 knockdown and ART significantly inhibited tumor growth. CONCLUSION: EFNA3 could be a valuable prognostic factor in CM.

Uveal melanoma immunogenomics predict immunotherapy resistance and susceptibility.

Immune checkpoint inhibition has shown success in treating metastatic cutaneous melanoma but has limited efficacy against metastatic uveal melanoma, a rare variant arising from the immune privileged eye. To better understand this resistance, we comprehensively profile 100 human uveal melanoma metastases using clinicogenomics, transcriptomics, and tumor infiltrating lymphocyte potency assessment. We find that over half of these metastases harbor tumor infiltrating lymphocytes with potent autologous tumor specificity, despite low mutational burden and resistance to prior immunotherapies. However, we observe strikingly low intratumoral T cell receptor clonality within the tumor microenvironment even after prior immunotherapies. To harness these quiescent tumor infiltrating lymphocytes, we develop a transcriptomic biomarker to enable in vivo identification and ex vivo liberation to counter their growth suppression. Finally, we demonstrate that adoptive transfer of these transcriptomically selected tumor infiltrating lymphocytes can promote tumor immunity in patients with metastatic uveal melanoma when other immunotherapies are incapable.

Predicting immunotherapy response in melanoma using a novel tumor immunological phenotype-related gene index.

Introduction: Melanoma is a highly aggressive and recurrent form of skin cancer, posing challenges in prognosis and therapy prediction. Methods: In this study, we developed a novel TIPRGPI consisting of 20 genes using Univariate Cox regression and the LASSO algorithm. The high and low-risk groups based on TIPRGPI exhibited distinct mutation profiles, hallmark pathways, and immune cell infiltration in the tumor microenvironment. Results: Notably, significant differences in tumor immunogenicity and TIDE were observed between the risk groups, suggesting a better response to immune checkpoint blockade therapy in the low-TIPRGPI group. Additionally, molecular docking predicted 10 potential drugs that bind to the core target, PTPRC, of the TIPRGPI signature. Discussion: Our findings highlight the reliability of TIPRGPI as a prognostic signature and its potential application in risk classification, immunotherapy response prediction, and drug candidate identification for melanoma treatment. The "TIP genes" guided strategy presented in this study may have implications beyond melanoma and could be applied to other cancer types.

circTADA2A inhibited SLC38A1 expression and suppresses melanoma progression through the prevention of CNBP trans-activation.

BACKGROUND: CircTADA2A has been demonstrated to play critical roles in the occurrence and development of human cancer. However, the expression pattern and biological mechanisms of circTADA2A in melanoma remains largely unknown. METHODS: CircTADA2A were detected by quantitative real-time RT-PCR (qRT-PCR) and validated by Sanger sequencing. Function of circTADA2A and its protein partner in melanoma cells was investigated using RNA interference and overexpression assays. Interaction of circTADA2A, CCHC-type zinc finger nucleic acid binding protein (CNBP) and solute carrier family 38 member 1 (SLC38A1) was confirmed by RNA immunoprecipitation, RNA pull-down, and dual-luciferase reporter assay. The expression of genes and proteins were detected by qRT-PCR and western blot assays. RESULTS: Data from the investigation showed that a novel circRNA (circTADA2A, hsa_circ_0043278) was markedly downregulated in melanoma cells. Functionally, circTADA2A repressed cell proliferation, migration, invasion in melanoma cells. Mechanistically, circTADA2A interacted with CNBP, acting to suppress the binding of CNBP to the SLC38A1 promoter and subsequently restrained SLC38A1 transcription, which resulting in repression of melanoma progression. CONCLUSIONS: CircTADA2A suppresses melanoma progression by regulating CNBP/SLC38A1 axis, indicating a potential therapeutic target in melanoma.

Genetic predisposition to childhood obesity does not influence the risk of developing skin cancer in adulthood.

The relationship between body mass index (BMI) and melanoma and other skin cancers remains unclear. The objective of this study was to employ the Mendelian randomization (MR) approach to evaluate the effects of genetically predicted childhood adiposity on the risk of developing skin cancer later in life. Two-sample MR analyses were conducted using summary data from genome-wide association study (GWAS) meta-analyses of childhood BMI, melanoma, cutaneous squamous cell carcinoma (cSCC), and basal cell carcinoma (BCC). We used the inverse-variance-weighted (IVW) methods to obtain a pooled estimate across all genetic variants for childhood BMI. We performed multiple sensitivity analyses to evaluate the potential influence of various assumptions on our findings. We found no evidence that genetically predicted childhood BMI was associated with risks of developing melanoma, cSCC, or BCC in adulthood (OR, 95% CI: melanoma: 1.02 (0.93-1.13), cSCC 0.94 (0.79-1.11), BCC 0.97 (0.84-1.12)). Our findings do not support the conclusions from observational studies that childhood BMI is associated with increased risks of melanoma, cSCC, or BCC in adulthood. Intervening on childhood adiposity will not reduce the risk of common skin cancers later in life.

Uveal melanoma cell lines Mel270 and 92.1 exhibit a mesenchymal phenotype and sensitivity to the cytostatic effects of transforming growth factor beta in vitro.

Purpose: Uveal melanoma (UM) is a deadly cancer with limited therapeutic options. At advanced stages, UM cells metastasize almost exclusively into the liver, where targeting metastatic UM cells remain a clinical challenge. Transforming growth factor beta (TGF-ß) exhibits a functional duality in cancer, with one arm limiting tumor growth at an early stage and the second arm promoting metastasis at an advanced stage, notably by inducing an epithelial-to-mesenchymal transition. Thus, we hypothesized that targeting the TGF-ß pathway could be relevant in the treatment of metastatic UM. Methods: In this study, we first characterized the pseudoepithelial/mesenchymal phenotype of UM cell lines Mel270 and 92.1. We then treated the cell lines with TGF-ß to evaluate their responsiveness to the cytokine and to characterize the functional impact of TGF-ß on their cell viability. Results: The results demonstrated, first, that the UM cell lines exhibited a mesenchymal phenotype and responded to TGF-ß treatment in vitro and, second, that TGF-ß promoted a cytostatic effect on the UM cell lines. Conclusions: Our findings indicate that UM cells are sensitive to the two arms of TGF-ß signaling, which suggests that targeting the TGF-ß pathway could be challenging in UM and would require a precise selection of patients in which only the prometastatic arm of TGF-ß is activated.

The Pediatric and Young Adult Choroidal and Ciliary Body Melanoma Genetic Study, A Survey by the European Ophthalmic Oncology Group.

Purpose: To explore the genetic background of choroidal and ciliary body melanoma among children and young adults, with special focus on BAP1 germline variants in this age group. Methods: Patients under the age of 25 and with confirmed choroidal or ciliary body melanoma were included in this retrospective, multicenter observational study. Nuclear BAP1 immunopositivity was used to evaluate the presence of functional BAP1 in the tumor. Next-generation sequencing using Ion Torrent platform was used to determine pathogenic variants of BAP1, EIF1AX, SF3B1, GNAQ and GNA11 and chromosome 3 status in the tumor or in DNA extracted from blood or saliva. Survival was analyzed using Kaplan-Meier estimates. Results: The mean age at diagnosis was 17 years (range 5.0-24.8). A germline BAP1 pathogenic variant was identified in an 18-year-old patient, and a somatic variant, based mainly on immunohistochemistry, in 13 (42%) of 31 available specimens. One tumor had a somatic SF3B1 pathogenic variant. Disomy 3 and the absence of a BAP1 pathogenic variant in the tumor predicted the longest metastasis-free survival. Males showed longer metastasis-free survival than females (P = 0.018). Conclusions: We did not find a stronger-than-average BAP1 germline predisposition for choroidal and ciliary body melanoma among children and young adults compared to adults. Males had a more favorable survival and disomy 3, and the absence of a BAP1 mutation in the tumor tissue predicted the most favorable metastasis-free survival. A BAP1 germline pathogenic variant was identified in one patient (1%), and a somatic variant based mainly on immunohistochemistry in 13 (42%).

What Is the Timing and Role of Targeted Therapy in Metastatic Melanoma?

ABSTRACT: Melanoma is the most lethal cutaneous malignancy worldwide. The last 15 years have ushered in several regulatory approvals that have dramatically altered the landscape of treatment options for patients with melanoma. Many patients with melanoma harbor activating mutations in the BRAF proto-oncogene, a key component of the mitogen-activated protein kinase (MAPK) intracellular signaling pathway. Therapies targeting BRAF have led to remarkable improvements in both response rates and survival in patients with metastatic disease. In parallel with these developments in MAPK-targeted therapy has been the clinical development of immune checkpoint inhibitors, which also have improved response rates and survival in patients with metastatic disease including randomized trials compared with MAPK-targeted therapy in patients with advanced, BRAF-mutant melanoma. Immune checkpoint inhibitors have become the preferred first-line standard-of-care treatment for patients with newly diagnosed metastatic disease in patients irrespective of BRAF mutational status. Given these developments, it is now less clear how to optimize the use of MAPK-targeted therapy regarding treatment setting and in sequence with immune checkpoint inhibitor.

The outcome in patients with BRAF-mutated metastatic melanoma treated with anti-programmed death receptor-1 monotherapy or targeted therapy in the real-world setting.

BACKGROUND: Immunotherapy and targeted therapy are currently two alternative backbones in the therapy of BRAF-mutated malignant melanoma. However, predictive biomarkers that would help with treatment selection are lacking. METHODS: This retrospective study investigated outcomes of anti-programmed death receptor-1 monotherapy and targeted therapy in the first-line setting in patients with metastatic BRAF-mutated melanoma, focusing on clinical and laboratory parameters associated with treatment outcome. RESULTS: Data from 174 patients were analysed. The median progression-free survival (PFS) was 17.0 months (95% CI; 8-39) and 12.5 months (95% CI; 9-14.2) for immunotherapy and targeted therapy, respectively. The 3-year PFS rate was 39% for immunotherapy and 25% for targeted therapy. The objective response rate was 72% and 51% for targeted therapy and immunotherapy. The median overall (OS) survival for immunotherapy has not been reached and was 23.6 months (95% CI; 16.1-38.2) for targeted therapy, with a 3-year survival rate of 63% and 40%, respectively. In a univariate analysis, age < 70 years, a higher number of metastatic sites, elevated serum LDH and a neutrophil-lymphocyte ratio above the cut-off value were associated with inferior PFS regardless of the therapy received, but only serum LDH level and the presence of lung metastases remained significant predictors of PFS in a multivariate analysis. CONCLUSIONS: Present real-world data document the high effectiveness of immunotherapy and targeted therapy. Although targeted therapy had higher response rates, immunotherapy improved PFS and OS. While the prognostic value of LDH was confirmed, the potential use of blood cell count-derived parameters to predict outcomes needs further investigation.

Dose optimization of an adjuvanted peptide-based personalized neoantigen melanoma vaccine.

The advancements in next-generation sequencing have made it possible to effectively detect somatic mutations, which has led to the development of personalized neoantigen cancer vaccines that are tailored to the unique variants found in a patient's cancer. These vaccines can provide significant clinical benefit by leveraging the patient's immune response to eliminate malignant cells. However, determining the optimal vaccine dose for each patient is a challenge due to the heterogeneity of tumors. To address this challenge, we formulate a mathematical dose optimization problem based on a previous mathematical model that encompasses the immune response cascade produced by the vaccine in a patient. We propose an optimization approach to identify the optimal personalized vaccine doses, considering a fixed vaccination schedule, while simultaneously minimizing the overall number of tumor and activated T cells. To validate our approach, we perform in silico experiments on six real-world clinical trial patients with advanced melanoma. We compare the results of applying an optimal vaccine dose to those of a suboptimal dose (the dose used in the clinical trial and its deviations). Our simulations reveal that an optimal vaccine regimen of higher initial doses and lower final doses may lead to a reduction in tumor size for certain patients. Our mathematical dose optimization offers a promising approach to determining an optimal vaccine dose for each patient and improving clinical outcomes.