Identifying Ferroptosis Inducers, HDAC, and RTK Inhibitor Sensitivity in Melanoma Subtypes through Unbiased Drug Target Prediction.

The utilization of PD1 and CTLA4 inhibitors has revolutionized the treatment of malignant melanoma (MM). However, resistance to targeted and immune-checkpoint-based therapies still poses a significant problem. Here we mine large scale MM proteogenomic data integrating it with MM cell line dependency screen, and drug sensitivity data to identify druggable targets and forecast treatment efficacy and resistance. Leveraging protein profiles from established MM subtypes and molecular structures of 82 cancer treatment drugs, we identified nine candidate hub proteins, mTOR, FYN, PIK3CB, EGFR, MAPK3, MAP4K1, MAP2K1, SRC and AKT1, across five distinct MM subtypes. These proteins serve as potential drug targets applicable to one or multiple MM subtypes. By analyzing transcriptomic data from 48 publicly accessible melanoma cell lines sourced from Achilles and CRISPR dependency screens, we forecasted 162 potentially targetable genes. We also identified genetic resistance in 260 genes across at least one melanoma subtype. In addition, we employed publicly available compound sensitivity data (Cancer Therapeutics Response Portal, CTRPv2) on the cell lines to assess the correlation of compound effectiveness within each subtype. We have identified 20 compounds exhibiting potential drug impact in at least one melanoma subtype. Remarkably, employing this unbiased approach, we have uncovered compounds targeting ferroptosis, that demonstrate a striking 30x fold difference in sensitivity among different subtypes. This implies that the proteogenomic classification of melanoma has the potential to predict sensitivity to ferroptosis compounds. Our results suggest innovative and novel therapeutic strategies by stratifying melanoma samples through proteomic profiling, offering a spectrum of novel therapeutic interventions and prospects for combination therapy. Highlights: (1) Proteogenomic subtype classification can define the landscape of genetic dependencies in melanoma (2) Nine proteins from molecular subtypes were identified as potential drug targets for specified MM patients (3) 20 compounds identified that show potential effectiveness in at least one melanoma subtype (4) Proteogenomics can predict specific ferroptosis inducers, HDAC, and RTK Inhibitor sensitivity in melanoma subtypes.

Development and validation of time-to-event models to predict metastatic recurrence of localized cutaneous melanoma.

BACKGROUND: The recent expansion of immunotherapy for stage IIB/IIC melanoma highlights a growing clinical need to identify patients at high risk of metastatic recurrence and, therefore, most likely to benefit from this therapeutic modality. OBJECTIVE: To develop time-to-event risk prediction models for melanoma metastatic recurrence. METHODS: Patients diagnosed with stage I/II primary cutaneous melanoma between 2000 and 2020 at Mass General Brigham and Dana-Farber Cancer Institute were included. Melanoma recurrence date and type were determined by chart review. Thirty clinicopathologic factors were extracted from electronic health records. Three types of time-to-event machine-learning models were evaluated internally and externally in the distant versus locoregional/nonrecurrence prediction. RESULTS: This study included 954 melanomas (155 distant, 163 locoregional, and 636 1:2 matched nonrecurrences). Distant recurrences were associated with worse survival compared to locoregional/nonrecurrences (HR: 6.21, P < .001) and to locoregional recurrences only (HR: 5.79, P < .001). The Gradient Boosting Survival model achieved the best performance (concordance index: 0.816; time-dependent AUC: 0.842; Brier score: 0.103) in the external validation. LIMITATIONS: Retrospective nature and cohort from one geography. CONCLUSIONS: These results suggest that time-to-event machine-learning models can reliably predict the metastatic recurrence from localized melanoma and help identify high-risk patients who are most likely to benefit from immunotherapy.

Unfolding the secrets of small cell lung cancer progression: Novel approaches and insights through rapid autopsies.

The understanding of small cell lung cancer (SCLC) biology has increased dramatically in recent years, but the processes that allow SCLC to progress rapidly remain poorly understood. Here, we advocate the integration of rapid autopsies and preclinical models into SCLC research as a comprehensive strategy with the potential to revolutionize current treatment paradigms.

Dual targeting of BCL-2 and MCL-1 in the presence of BAX breaks venetoclax resistance in human small cell lung cancer.

BACKGROUND: No targeted drugs are currently available against small cell lung cancer (SCLC). BCL-2 family members are involved in apoptosis regulation and represent therapeutic targets in many malignancies. METHODS: Expression of BCL-2 family members in 27 SCLC cell lines representing all known four SCLC molecular subtypes was assessed by qPCR, Western blot and mass spectrometry-based proteomics. BCL-2 and MCL-1 inhibition (venetoclax and S63845, respectively) was assessed by MTT assay and flow cytometry and in mice bearing human SCLC tumours. Drug interactions were calculated using the Combenefit software. Ectopic BAX overexpression was achieved by expression plasmids. RESULTS: The highest BCL-2 expression levels were detected in ASCL1- and POU2F3-driven SCLC cells. Although sensitivity to venetoclax was reflected by BCL-2 levels, not all cell lines responded consistently despite their high BCL-2 expression. MCL-1 overexpression and low BAX levels were both characteristic for venetoclax resistance in SCLC, whereas the expression of other BCL-2 family members did not affect therapeutic efficacy. Combination of venetoclax and S63845 resulted in significant, synergistic in vitro and in vivo anti-tumour activity and apoptosis induction in double-resistant cells; however, this was seen only in a subset with detectable BAX. In non-responding cells, ectopic BAX overexpression sensitised to venetoclax and S63845 and, furthermore, induced synergistic drug interaction. CONCLUSIONS: The current study reveals the subtype specificity of BCL-2 expression and sheds light on the mechanism of venetoclax resistance in SCLC. Additionally, we provide preclinical evidence that combined BCL-2 and MCL-1 targeting is an effective approach to overcome venetoclax resistance in high BCL-2-expressing SCLCs with intact BAX.

Newly identified form of phenotypic plasticity of cancer: immunogenic mimicry.

Cancer plasticity is now a recognized new hallmark of cancer which is due to disturbances of cell differentiation programs. It is manifested not only in various forms like the best-known epithelial-mesenchymal transition (EMT) but also in vasculogenic and megakaryocytic mimicries regulated by EMT-specific or less-specific transcription factors such as HIF1a or STAT1/2. Studies in the past decades provided ample data that cancer plasticity can be manifested also in the expression of a vast array of immune cell genes; best-known examples are PDL1/CD274, CD47, or IDO, and we termed it immunogenic mimicry (IGM). However, unlike other types of plasticities which are epigenetically regulated, expression of IGM genes are frequently due to gene amplifications. It is important that the majority of the IGM genes are regulated by interferons (IFNs) suggesting that their protein expressions are regulated by the immune microenvironment. Most of the IGM genes have been shown to be involved in immune escape of cancers broadening the repertoire of these mechanisms and offering novel targets for immunotherapeutics.

Prognostic importance of biomarkers associated with haemostatic, vascular and endothelial disturbances in acute coronary syndrome patients in relation to kidney function.

BACKGROUND: Patients with kidney failure have a high risk for cardiovascular events. We aimed to evaluate the prognostic importance of selected biomarkers related to haemostasis, endothelial function, and vascular regulation in patients with acute coronary syndrome (ACS), and to study whether this association differed in patients with renal dysfunction. METHODS: Plasma was collected in 1370 ACS patients included between 2008 and 2015. Biomarkers were analysed using a Proximity Extension Assay and a Multiple Reaction Monitoring mass spectrometry assay. To reduce multiplicity, biomarkers correlating with eGFR were selected a priori among 36 plasma biomarkers reflecting endothelial and vascular function, and haemostasis. Adjusted Cox regression were used to study their association with the composite outcome of myocardial infarction, ischemic stroke, heart failure or death. Interaction with eGFR strata above or below 60 ml/min/1.73 m2 was tested. RESULTS: Tissue factor, proteinase-activated receptor, soluble urokinase plasminogen activator surface receptor (suPAR), thrombomodulin, adrenomedullin, renin, and angiotensinogen correlated inversely with eGFR and were selected for the Cox regression. Mean follow-up was 5.2 years during which 428 events occurred. Adrenomedullin, suPAR, and renin were independently associated with the composite outcome. Adrenomedullin showed interaction with eGFR strata (p = 0.010) and was associated with increased risk (HR 1.88; CI 1.44-2.45) only in patients with eGFR ≥60 ml/min/ 1.73 m2. CONCLUSIONS: Adrenomedullin, suPAR, and renin were associated with the composite outcome in all. Adrenomedullin, involved in endothelial protection, showed a significant interaction with renal function and outcome, and was associated with the composite outcome only in patients with preserved kidney function.

Prediction of early-stage melanoma recurrence using clinical and histopathologic features.

Prognostic analysis for early-stage (stage I/II) melanomas is of paramount importance for customized surveillance and treatment plans. Since immune checkpoint inhibitors have recently been approved for stage IIB and IIC melanomas, prognostic tools to identify patients at high risk of recurrence have become even more critical. This study aims to assess the effectiveness of machine-learning algorithms in predicting melanoma recurrence using clinical and histopathologic features from Electronic Health Records (EHRs). We collected 1720 early-stage melanomas: 1172 from the Mass General Brigham healthcare system (MGB) and 548 from the Dana-Farber Cancer Institute (DFCI). We extracted 36 clinicopathologic features and used them to predict the recurrence risk with supervised machine-learning algorithms. Models were evaluated internally and externally: (1) five-fold cross-validation of the MGB cohort; (2) the MGB cohort for training and the DFCI cohort for testing independently. In the internal and external validations, respectively, we achieved a recurrence classification performance of AUC: 0.845 and 0.812, and a time-to-event prediction performance of time-dependent AUC: 0.853 and 0.820. Breslow tumor thickness and mitotic rate were identified as the most predictive features. Our results suggest that machine-learning algorithms can extract predictive signals from clinicopathologic features for early-stage melanoma recurrence prediction, which will enable the identification of patients that may benefit from adjuvant immunotherapy.

In-depth proteomic analysis reveals unique subtype-specific signatures in human small-cell lung cancer.

BACKGROUND: Small-cell lung cancer (SCLC) molecular subtypes have been primarily characterized based on the expression pattern of the following key transcription regulators: ASCL1 (SCLC-A), NEUROD1 (SCLC-N), POU2F3 (SCLC-P) and YAP1 (SCLC-Y). Here, we investigated the proteomic landscape of these molecular subsets with the aim to identify novel subtype-specific proteins of diagnostic and therapeutic relevance. METHODS: Pellets and cell media of 26 human SCLC cell lines were subjected to label-free shotgun proteomics for large-scale protein identification and quantitation, followed by in-depth bioinformatic analyses. Proteomic data were correlated with the cell lines' phenotypic characteristics and with public transcriptomic data of SCLC cell lines and tissues. RESULTS: Our quantitative proteomic data highlighted that four molecular subtypes are clearly distinguishable at the protein level. The cell lines exhibited diverse neuroendocrine and epithelial-mesenchymal characteristics that varied by subtype. A total of 367 proteins were identified in the cell pellet and 34 in the culture media that showed significant up- or downregulation in one subtype, including known druggable proteins and potential blood-based markers. Pathway enrichment analysis and parallel investigation of transcriptomics from SCLC cell lines outlined unique signatures for each subtype, such as upregulated oxidative phosphorylation in SCLC-A, DNA replication in SCLC-N, neurotrophin signalling in SCLC-P and epithelial-mesenchymal transition in SCLC-Y. Importantly, we identified the YAP1-driven subtype as the most distinct SCLC subgroup. Using sparse partial least squares discriminant analysis, we identified proteins that clearly distinguish four SCLC subtypes based on their expression pattern, including potential diagnostic markers for SCLC-Y (e.g. GPX8, PKD2 and UFO). CONCLUSIONS: We report for the first time, the protein expression differences among SCLC subtypes. By shedding light on potential subtype-specific therapeutic vulnerabilities and diagnostic biomarkers, our results may contribute to a better understanding of SCLC biology and the development of novel therapies.

Lipid Metabolic Reprogramming Extends beyond Histologic Tumor Demarcations in Operable Human Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest malignancies and potentially curable only with radical surgical resection at early stages. The tumor microenvironment has been shown to be central to the development and progression of PDAC. A better understanding of how early human PDAC metabolically communicates with its environment and differs from healthy pancreas could help improve PDAC diagnosis and treatment. Here we performed deep proteomic analyses from diagnostic specimens of operable, treatment-naïve PDAC patients (n = 14), isolating four tissue compartments by laser-capture microdissection: PDAC lesions, tumor-adjacent but morphologically benign exocrine glands, and connective tissues neighboring each of these compartments. Protein and pathway levels were compared between compartments and with control pancreatic proteomes. Selected targets were studied immunohistochemically in the 14 patients and in additional tumor microarrays, and lipid deposition was assessed by nonlinear label-free imaging (n = 16). Widespread downregulation of pancreatic secretory functions was observed, which was paralleled by high cholesterol biosynthetic activity without prominent lipid storage in the neoplastic cells. Stromal compartments harbored ample blood apolipoproteins, indicating abundant microvasculature at the time of tumor removal. The features best differentiating the tumor-adjacent exocrine tissue from healthy control pancreas were defined by upregulation of proteins related to lipid transport. Importantly, histologically benign exocrine regions harbored the most significant prognostic pathways, with proteins involved in lipid transport and metabolism, such as neutral cholesteryl ester hydrolase 1, associating with shorter survival. In conclusion, this study reveals prognostic molecular changes in the exocrine tissue neighboring pancreatic cancer and identifies enhanced lipid transport and metabolism as its defining features. SIGNIFICANCE: In clinically operable pancreatic cancer, regions distant from malignant cells already display proteomic changes related to lipid transport and metabolism that affect prognosis and may be pharmacologically targeted.

Expression patterns and prognostic relevance of subtype-specific transcription factors in surgically resected small-cell lung cancer: an international multicenter study.

The tissue distribution and prognostic relevance of subtype-specific proteins (ASCL1, NEUROD1, POU2F3, YAP1) present an evolving area of research in small-cell lung cancer (SCLC). The expression of subtype-specific transcription factors and P53 and RB1 proteins were measured by immunohistochemistry (IHC) in 386 surgically resected SCLC samples. Correlations between subtype-specific proteins and in vitro efficacy of various therapeutic agents were investigated by proteomics and cell viability assays in 26 human SCLC cell lines. Besides SCLC-A (ASCL1-dominant), SCLC-AN (combined ASCL1/NEUROD1), SCLC-N (NEUROD1-dominant), and SCLC-P (POU2F3-dominant), IHC and cluster analyses identified a quadruple-negative SCLC subtype (SCLC-QN). No unique YAP1-subtype was found. The highest overall survival rates were associated with non-neuroendocrine subtypes (SCLC-P and SCLC-QN) and the lowest with neuroendocrine subtypes (SCLC-A, SCLC-N, SCLC-AN). In univariate analyses, high ASCL1 expression was associated with poor prognosis and high POU2F3 expression with good prognosis. Notably, high ASCL1 expression influenced survival outcomes independently of other variables in a multivariate model. High POU2F3 and YAP1 protein abundances correlated with sensitivity and resistance to standard-of-care chemotherapeutics, respectively. Specific correlation patterns were also found between the efficacy of targeted agents and subtype-specific protein abundances. In conclusion, we investigated the clinicopathological relevance of SCLC molecular subtypes in a large cohort of surgically resected specimens. Differential IHC expression of ASCL1, NEUROD1, and POU2F3 defines SCLC subtypes. No YAP1-subtype can be distinguished by IHC. High POU2F3 expression is associated with improved survival in a univariate analysis, whereas elevated ASCL1 expression is an independent negative prognosticator. Proteomic and cell viability assays of human SCLC cell lines revealed distinct vulnerability profiles defined by transcription regulators. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Non-Invasive, Topical Sampling of Potential, Low-Molecular Weight, Skin Cancer Biomarkers: A Study on Healthy Volunteers.

Monitoring of low-molecular weight cancer biomarkers, such as tryptophan (Trp) and its derivative kynurenine (Kyn), might be advantageous to non-invasive skin cancer detection. Thus, we assessed several approaches of topical sampling of Trp and Kyn, in relation to phenylalanine (Phe) and tyrosine (Tyr), on the volar forearm of six healthy volunteers. The sampling was performed with three hydrogels (made of agarose or/and chitosan), hydrated starch films, cotton swabs, and tape stripping. The biomarkers were successfully sampled by all approaches, but the amount of collected Kyn was low, 20 ± 10 pmol/cm2. Kyn quantification was below LOQ, and thus, it was detected only in 20% of topical samples. To mitigate variability problems of absolute amounts of sampled amino acids, Tyr/Trp, Phe/Trp, and Phe/Tyr ratios were assessed, proving reduced inter-individual variation from 79 to 45% and intra-individual variation from 42 to 21%. Strong positive correlation was found between Phe and Trp, pointing to the Phe/Trp ratio (being in the 1.0-2.0 range, at 95% confidence) being least dependent on sampling materials, approaches, and sweating. This study leads to conclusion that due to the difficulty in quantifying less abundant Kyn, and thus the Trp/Kyn ratio, the Phe/Trp ratio might be a possible, alternative biomarker for detecting skin cancers.

Novel protein markers of androgen activity in humans: proteomic study of plasma from young chemically castrated men.

BACKGROUND: Reliable biomarkers of androgen activity in humans are lacking. The aim of this study was, therefore, to identify new protein markers of biological androgen activity and test their predictive value in relation to low vs normal testosterone values and some androgen deficiency linked pathologies. METHODS: Blood samples from 30 healthy GnRH antagonist treated males were collected at three time points: (1) before GnRH antagonist administration; (2) 3 weeks later, just before testosterone undecanoate injection, and (3) after additional 2 weeks. Subsequently, they were analyzed by mass spectrometry to identify potential protein biomarkers of testosterone activity. Levels of proteins most significantly associated with testosterone fluctuations were further tested in a cohort of 75 hypo- and eugonadal males suffering from infertility. Associations between levels of those markers and cardiometabolic parameters, bone mineral density as well as androgen receptor (AR) CAG repeat lengths, were explored. RESULTS: Using receiver operating characteristic analysis, 4-hydroxyphenylpyruvate dioxygenase (4HPPD), insulin-like growth factor-binding protein 6 (IGFBP6), and fructose-bisphosphate aldolase (ALDOB), as well as a Multi Marker Algorithm, based on levels of 4HPPD and IGFBP6, were shown to be best predictors of low (<8 nmol/l) vs normal (>12 nmol/l) testosterone. They were also more strongly associated with metabolic syndrome and diabetes than testosterone levels. Levels of ALDOB and 4HPPD also showed association with AR CAG repeat lengths. CONCLUSIONS: We identified potential new protein biomarkers of testosterone action. Further investigations to elucidate their clinical potential are warranted. FUNDING: The work was supported by ReproUnion2.0 (grant no. 20201846), which is funded by the Interreg V EU program.

Although it is best known for its role in developing male sex organs and maintaining sexual function, the hormone testosterone is important for many parts of the human body. A deficiency can cause an increased risk of serious conditions such as diabetes, cancer and osteoporosis. Testosterone deficiency can develop due to disease or age-related changes, and men affected by this can be given supplements of this hormone to restore normal levels. The most common way to test for testosterone deficiency is by measuring the concentration of the hormone in the blood. However, this does not accurately reflect the activity of the hormone in the body. This may lead to men who need more testosterone not receiving enough, and to others being unnecessarily treated. Several factors may lead to discrepancy between testosterone concentration in blood and its physiological activity. One of the most common is obesity. Additionally, certain genetic factors, which cannot be controlled for yet, regulate sensitivity to this hormone: some people do well at low levels, while others need high concentrations to be healthy. Therefore, to improve the diagnosis of testosterone deficiency it is necessary to identify biological markers whose levels act as a proxy for testosterone activity. Giwercman, Sahlin et al. studied the levels of a large number of proteins in the blood of 30 young men before and after blocking testosterone production. The analysis found three proteins whose concentrations changed significantly after testosterone deprivation. Giwercman, Sahlin et al. then validated these markers for testosterone deficiency by checking the levels of the three proteins in a separate group of 75 men with fertility problems. The results also showed that the three protein markers were better at predicting diabetes and metabolic syndrome than testosterone levels alone. These newly discovered markers could be used to create a test for measuring testosterone activity. This could help to identify deficiencies and finetune the amount of supplementary hormone given to men as treatment. However, further research is needed to understand the clinical value of such a test in men, as well as women and children.

The Prognostic Relevance of PMCA4 Expression in Melanoma: Gender Specificity and Implications for Immune Checkpoint Inhibition.

PMCA4 is a critical regulator of Ca2+ homeostasis in mammalian cells. While its biological and prognostic relevance in several cancer types has already been demonstrated, only preclinical investigations suggested a metastasis suppressor function in melanoma. Therefore, we studied the expression pattern of PMCA4 in human skin, nevus, as well as in primary and metastatic melanoma using immunohistochemistry. Furthermore, we analyzed the prognostic power of PMCA4 mRNA levels in cutaneous melanoma both at the non-metastatic stage as well as after PD-1 blockade in advanced disease. PMCA4 localizes to the plasma membrane in a differentiation dependent manner in human skin and mucosa, while nevus cells showed no plasma membrane staining. In contrast, primary cutaneous, choroidal and conjunctival melanoma cells showed specific plasma membrane localization of PMCA4 with a wide range of intensities. Analyzing the TCGA cohort, PMCA4 mRNA levels showed a gender specific prognostic impact in stage I-III melanoma. Female patients with high transcript levels had a significantly longer progression-free survival. Melanoma cell specific PMCA4 protein expression is associated with anaplasticity in melanoma lung metastasis but had no impact on survival after lung metastasectomy. Importantly, high PMCA4 transcript levels derived from RNA-seq of cutaneous melanoma are associated with significantly longer overall survival after PD-1 blockade. In summary, we demonstrated that human melanoma cells express PMCA4 and PMCA4 transcript levels carry prognostic information in a gender specific manner.

Landscape of surfaceome and endocytome in human glioma is divergent and depends on cellular spatial organization.

Therapeutic strategies directed at the tumor surfaceome (TS), including checkpoint inhibitor blocking antibodies, antibody drug conjugates (ADCs), and chimeric antigen receptor T (CAR-T) cells, provide a new armament to fight cancer. However, a remaining bottleneck is the lack of strategies to comprehensively interrogate patient tumors for potential TS targets. Here, we have developed a platform (tumor surfaceome mapping [TS-MAP]) integrated with a newly curated TS classifier (SURFME) that allows profiling of primary 3D cultures and intact patient glioma tumors with preserved tissue architecture. Moreover, TS-MAP specifically identifies proteins capable of endocytosis as tractable targets for ADCs and other modalities requiring toxic payload internalization. In high-grade gliomas that remain among the most aggressive forms of cancer, we show that cellular spatial organization (2D vs. 3D) fundamentally transforms the surfaceome and endocytome (e.g., integrins, proteoglycans, semaphorins, and cancer stem cell markers) with general implications for target screening approaches, as exemplified by an ADC targeting EGFR. The TS-MAP platform was further applied to profile the surfaceome and endocytome landscape in a cohort of freshly resected gliomas. We found a highly diverse TS repertoire between patient tumors, not directly associated with grade and histology, which highlights the need for individualized approaches. Our data provide additional layers of understanding fundamental to the future development of immunotherapy strategies, as well as procedures for proteomics-based target identification and selection. The TS-MAP platform should be widely applicable in efforts aiming at a better understanding of how to harness the TS for personalized immunotherapy.

Organ Specific Copy Number Variations in Visceral Metastases of Human Melanoma.

Malignant melanoma is one of the most aggressive skin cancers with high potential of visceral dissemination. Since the information about melanoma genomics is mainly based on primary tumors and lymphatic or skin metastases, an autopsy-based visceral metastasis biobank was established. We used copy number variation arrays (N = 38 samples) to reveal organ specific alterations. Results were partly completed by proteomic analysis. A significant increase of high-copy number gains was found in an organ-specific manner, whereas copy number losses were predominant in brain metastases, including the loss of numerous DNA damage response genes. Amplification of many immune genes was also observed, several of them are novel in melanoma, suggesting that their ectopic expression is possibly underestimated. This "immunogenic mimicry" was exclusive for lung metastasis. We also provided evidence for the possible autocrine activation of c-MET, especially in brain and lung metastases. Furthermore, frequent loss of 9p21 locus in brain metastases may predict higher metastatic potential to this organ. Finally, a significant correlation was observed between BRAF gene copy number and mutant allele frequency, mainly in lung metastases. All of these events may influence therapy efficacy in an organ specific manner, which knowledge may help in alleviating difficulties caused by resistance.

An Observational Study on the Molecular Profiling of Primary Melanomas Reveals a Progression Dependence on Mitochondrial Activation.

Melanoma in advanced stages is one of the most aggressive tumors and the deadliest of skin cancers. To date, the histopathological staging focuses on tumor thickness, and clinical staging is a major estimate of the clinical behavior of primary melanoma. Here we report on an observational study with in-depth molecular profiling at the protein level including post-translational modifications (PTMs) on eleven primary tumors from melanoma patients. Global proteomics, phosphoproteomics, and acetylomics were performed on each sample. We observed an up-regulation of key mitochondrial functions, including the mitochondrial translation machinery and the down-regulation of structural proteins involved in cell adhesion, the cytoskeleton organization, and epidermis development, which dictates the progression of the disease. Additionally, the PTM level pathways related to RNA processing and transport, as well as chromatin organization, were dysregulated in relation to the progression of melanoma. Most of the pathways dysregulated in this cohort were enriched in genes differentially expressed at the transcript level when similar groups are compared or metastasis to primary melanomas. At the genome level, we found significant differences in the mutation profiles between metastatic and primary melanomas. Our findings also highlighted sex-related differences in the molecular profiles. Remarkably, primary melanomas in women showed higher levels of antigen processing and presentation, and activation of the immune system response. Our results provide novel insights, relevant for developing personalized precision treatments for melanoma patients.

Deep Proteomic Analysis on Biobanked Paraffine-Archived Melanoma with Prognostic/Predictive Biomarker Read-Out.

The discovery of novel protein biomarkers in melanoma is crucial. Our introduction of formalin-fixed paraffin-embedded (FFPE) tumor protocol provides new opportunities to understand the progression of melanoma and open the possibility to screen thousands of FFPE samples deposited in tumor biobanks and available at hospital pathology departments. In our retrospective biobank pilot study, 90 FFPE samples from 77 patients were processed. Protein quantitation was performed by high-resolution mass spectrometry and validated by histopathologic analysis. The global protein expression formed six sample clusters. Proteins such as TRAF6 and ARMC10 were upregulated in clusters with enrichment for shorter survival, and proteins such as AIFI1 were upregulated in clusters with enrichment for longer survival. The cohort's heterogeneity was addressed by comparing primary and metastasis samples, as well comparing clinical stages. Within immunotherapy and targeted therapy subgroups, the upregulation of the VEGFA-VEGFR2 pathway, RNA splicing, increased activity of immune cells, extracellular matrix, and metabolic pathways were positively associated with patient outcome. To summarize, we were able to (i) link global protein expression profiles to survival, and they proved to be an independent prognostic indicator, as well as (ii) identify proteins that are potential predictors of a patient's response to immunotherapy and targeted therapy, suggesting new opportunities for precision medicine developments.

Mapping the Melanoma Plasma Proteome (MPP) Using Single-Shot Proteomics Interfaced with the WiMT Database.

Plasma analysis by mass spectrometry-based proteomics remains a challenge due to its large dynamic range of 10 orders in magnitude. We created a methodology for protein identification known as Wise MS Transfer (WiMT). Melanoma plasma samples from biobank archives were directly analyzed using simple sample preparation. WiMT is based on MS1 features between several MS runs together with custom protein databases for ID generation. This entails a multi-level dynamic protein database with different immunodepletion strategies by applying single-shot proteomics. The highest number of melanoma plasma proteins from undepleted and unfractionated plasma was reported, mapping >1200 proteins from >10,000 protein sequences with confirmed significance scoring. Of these, more than 660 proteins were annotated by WiMT from the resulting ~5800 protein sequences. We could verify 4000 proteins by MS1t analysis from HeLA extracts. The WiMT platform provided an output in which 12 previously well-known candidate markers were identified. We also identified low-abundant proteins with functions related to (i) cell signaling, (ii) immune system regulators, and (iii) proteins regulating folding, sorting, and degradation, as well as (iv) vesicular transport proteins. WiMT holds the potential for use in large-scale screening studies with simple sample preparation, and can lead to the discovery of novel proteins with key melanoma disease functions.

Development of Novel VEGFR2 Inhibitors Originating from Natural Product Analogues with Antiangiogenic Impact.

A novel natural small molecule, voacangine (Voa), has been discovered as a potent antiangiogenic compound. Notably, Voa directly binds the kinase domain of the vascular endothelial growth factor receptor 2 (VEGFR2) and thereby inhibits downstream signaling. Herein, we developed synthetic small molecules based on the unique chemical structure of Voa that directly and specifically target and modulate the kinase activity of VEGFR2. Among these Voa structure analogues, Voa analogue 19 (V19) exhibited increased antiangiogenic potency against VEGF-induced VEGFR2 phosphorylation without cytotoxic effects. Moreover, treatment with V19 resulted in significant tumor cell death in a mouse xenograft model. In conclusion, this new VEGFR2 modulator, inspired from the rigid scaffold of a natural compound, Voa, is presented as a potent candidate in the development of new antiangiogenic agents.