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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.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 mTissue 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 mAdrenomedullin, 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.

Supplementary Data from 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.

The hippocampus is a primary region affected in Alzheimer’s disease (AD). Because AD postmortem brain tissue is not available prior to symptomatic stage, we lack understanding of early cellular pathogenic mechanisms. To address this issue, we examined the cellular origin and progression of AD pathogenesis in patient-based model systems including iPSC-derived brain cells transplanted into the mouse brain hippocampus. Notably, proteomic analysis of the graft enabled the identification of proteomic alterations in AD patient brain cells, associated with increased levels of β-sheet structures and Aβ42 peptides. Interestingly, the host cells surrounding the AD graft also presented alterations in cellular biological pathways. Furthermore, proteomic analysis across human iPSC-based models and human post-mortem hippocampal tissue projected coherent longitudinal cellular changes indicative of disease progression from early to end stage AD. Our data showcase patient-based models to study the cellular origin, progression, and prion-like spread of AD pathogenesis.Highlights-AD patient iPSC-derived brain cells survive in the hippocampus of immunodeficient mice 6 months post-transplantation.-Proteomic analysis of the grafts reveals profound alterations in cellular biological pathways in iPSC-derived hippocampal cells despite absence of senile plaques.-Proteomic alterations within transplanted AD iPSC-derived hippocampal cells are reminiscent of early/prodromal AD.-AD-grafted cells induce proteomic changes in host mouse cells.

Monitoring of low-molecular weight cancer biomarkers, suchas tryptophan (Trp) and its derivative kynurenine (Kyn), might beadvantageous to non-invasive skin cancer detection. Thus, we assessedseveral approaches of topical sampling of Trp and Kyn, in relation tophenylalanine (Phe) and tyrosine (Tyr), on the volar forearm of six healthyvolunteers. The sampling was performed with three hydrogels (made ofagarose or/and chitosan), hydrated starchfilms, cotton swabs, and tapestripping. The biomarkers were successfully sampled by all approaches, butthe amount of collected Kyn was low, 20 +/- 10 pmol/cm2.Kynquantification was below LOQ, and thus, it was detected only in 20% oftopical samples. To mitigate variability problems of absolute amounts ofsampled 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 foundbetween Phe and Trp, pointing to the Phe/Trp ratio (being in the 1.0-2.0 range, at 95% confidence) being least dependent onsampling materials, approaches, and sweating. This study leads to conclusion that due to the difficulty in quantifying less abundantKyn, and thus the Trp/Kyn ratio, the Phe/Trp ratio might be a possible, alternative biomarker for detecting skin cancers. Funding Agencies|Knowledge Foundation [20170058]

SummaryThe dynamics of more than 1900 mitochondrial proteins was explored through quantitative proteomics in 151 melanoma-related tissue samples of both surgical and autopsy origin. Dysregulation of mitochondrial pathways in primary tumors, metastases, and peritumoral tissues was correlated with age and survival of patients, as well as with tumor cell proliferation and the BRAF mutation status of the tumors. The outlined proteomic landscape confirmed the central role of a pathologically upregulated mitochondrial translation machinery and oxidative phosphorylation (OXPHOS) in the development, proliferation, and progression of melanomas. Our results from different melanoma cell lines confirmed our findings and we could document that treatments with selected OXPHOS inhibitors and antibiotics successfully impaired tumor cell proliferation. In addition, we provided proteomic evidence on the mechanism-of-action of the different treatments. These observations could contribute to the development of therapeutic approaches targeting the mitochondrial pathology in melanoma.TOC figureHighlightsMitochondrial proteome landscape outlined in 151 melanoma-related samplesMitochondrial Translation and OXPHOS impact disease severity and survivalBRAF V600E mutation correlates with upregulation of mitochondrial energy productionTargeting the mitochondrial OXPHOS and ribosomes impairs tumor cell proliferationTherapeutic opportunities complementary to the standard of care are proposedIn briefMitochondrial proteome profiling of melanomas reveals dysregulation in major metabolic pathways, suggesting a central role of the mitochondria within the development and progression of melanoma. Targeting mitochondrial pathways has the potential to impact the course of the disease, which provides opportunities for complementary drug interventions.

Huntington's disease is a neurodegenerative disorder caused by CAG expansions in the huntingtin (HTT) gene. Modelling Huntington's disease is challenging, as rodent and cellular models poorly recapitulate the disease as seen in ageing humans. To address this, we generated induced neurons through direct reprogramming of human skin fibroblasts, which retain age-dependent epigenetic characteristics. Huntington's disease induced neurons (HD-iNs) displayed profound deficits in autophagy, characterized by reduced transport of late autophagic structures from the neurites to the soma. These neurite-specific alterations in autophagy resulted in shorter, thinner and fewer neurites specifically in HD-iNs. CRISPRi-mediated silencing of HTT did not rescue this phenotype but rather resulted in additional autophagy alterations in control induced neurons, highlighting the importance of wild-type HTT in normal neuronal autophagy. In summary, our work identifies a distinct subcellular autophagy impairment in adult patient derived Huntington's disease neurons and provides a new rationale for future development of autophagy activation therapies.

Small cell lung cancer (SCLC) represents for 15% of all lung cancers. Although SCLC is initially highly sensitive to chemotherapy (CHT), almost all patients acquire resistance within the course of the disease. A promising class of compounds to overcome drug resistance comprises histone deacetylase inhibitors (HDACi), which have been shown to synergize with several CHT agents such as cisplatin. In this study, we addressed whether entinostat exhibits anti-cancer activity as single-therapy or in combination with CHT in SCLC. The efficacy of entinostat alone or in combination with CHT was determined using MTT-based viability assays and a mouse xenograft model. Synergistic drug interactions were evaluated using the combenefit software. Analysis of our previously published proteomic dataset comprised 1D annotation enrichment, DAVID functional annotation and ToppCluster analysis. Hypothetic mechanisms were also examined using ICP-MS and FACS-based cell cycle analysis. Class I HDAC inhibition by entinostat resulted in significantly stronger effects in neuroendocrine (NE) SCLC cell lines. Subsequent proteomic analysis revealed differentially expressed proteins and pathways that coincide with NE and non-NE characteristics. Since “platinum drug resistance” appeared in the corresponding pathway analysis, cell lines were exposed to cisplatin. A significant correlation was observed between entinostat and cisplatin responsiveness (r=0.8079, p=0.0003). With combination therapy, a strong synergism was detected in a subset of SCLC cell lines and validated in a mouse model. Importantly, similar effects were also found when entinostat was combined with etoposide, epirubicin or irinotecan. To address the underlying mechanism of the observed effects, another proteomic evaluation comparing cell lines with synergistic and additive features indicated changes in cell cycle regulation and DNA damage repair. Cell cycle analysis revealed similar distributions in control, entinostat and cisplatin conditions. However, synergistic cell lines displayed a shift from G1 to S-phase compared to additive cell lines upon combination treatment. Results from ICP-MS analysis revealed that although basal platinum levels were significantly higher in the synergistic subgroup, all SCLC cell lines showed increased intracellular platinum levels in the presence of entinostat. Here, we report that high NE expression profiles in SCLC cell lines are associated with increased sensitivity to HDAC inhibition and, moreover, that combinational therapy using entinostat and CHT results in strong synergism in a subset of SCLC cell lines featuring dysregulated cell cycle and DNA damage repair, according to proteomic analyses. Especially in SCLC, efficient therapeutic options in relapsed patients are missing, and deciphering the molecular basis of synergism between chemotherapy and entinostat might lead to more effective therapy for patients. Citation Format: Anna Schwendenwein, Kristiina Boettiger, Ildiko Kovacs, Nandor Barany, Christian Lang, Zsolt Megyesfalvi, Michael Grusch, Walter Berger, Christian Kowol, Melinda Rezeli, Konrad Hoetzenecker, Balazs Döme, Karin Schelch. Entinostat potentiates chemotherapeutic efficacy in small cell lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2230.

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 WileySons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

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.

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.

Simple Summary Malignant melanoma is the deadliest among the skin cancers. In the advanced stage, it shows extremely poor prognosis. The incidence and mortality of melanoma has been continuously increasing worldwide, primarily among the fair-skinned populations. To date, the best indicator for progression is the tumor thickness (Breslow). In this sense, there is a fundamental need to discover early indicators of disease progression. We believe that in-depth molecular profiling at the multiomic level of primary melanomas may find a signature that predicts disease progression toward metastasis. Here we report an observational study of a small primary melanoma cohort, in which in-depth molecular profiling was performed at the levels of protein expression, phosphorylation, and lysine acetylation. Our results provided evidence for dysregulation of pathways and biological processes at all molecular levels evaluated, in primary tumors from patients that ultimately developed a metastatic disease. Moreover, we provided evidence at the molecular level of the sex-related differences in the melanoma disease presentation. Abstract 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.