Oleuropein-driven reprogramming of the myeloid cell compartment to sensitise tumours to PD-1/PD-L1 blockade strategies.

BACKGROUND: Previous studies have shown that functional systemic immunity is required for the efficacy of PD-1/PD-L1 blockade immunotherapies in cancer. Hence, systemic reprogramming of immunosuppressive dysfunctional myeloid cells could overcome resistance to cancer immunotherapy. METHODS: Reprogramming of tumour-associated myeloid cells with oleuropein was studied by quantitative differential proteomics, phenotypic and functional assays in mice and lung cancer patients. Combinations of oleuropein and two different delivery methods of anti-PD-1 antibodies were tested in colorectal cancer tumour models and in immunotherapy-resistant lung cancer models. RESULTS: Oleuropein treatment reprogrammed monocytic and granulocytic myeloid-derived suppressor cells, and tumour-associated macrophages towards differentiation of immunostimulatory subsets. Oleuropein regulated major differentiation programmes associated to immune modulation in myeloid cells, which potentiated T cell responses and PD-1 blockade. PD-1 antibodies were delivered by two different strategies, either systemically or expressed within tumours using a self-amplifying RNA vector. Combination anti-PD-1 therapies with oleuropein increased tumour infiltration by immunostimulatory dendritic cells in draining lymph nodes, leading to systemic antitumour T cell responses. Potent therapeutic activities were achieved in colon cancer and lung cancer models resistant to immunotherapies, even leading to complete tumour regression. DISCUSSION: Oleuropein significantly improves the outcome of PD-1/PD-L1 blockade immunotherapy strategies by reprogramming myeloid cells.

Mapping the human brain proteome: opportunities, challenges, and clinical potential.

INTRODUCTION: Due to the segmented functions and complexity of the human brain, the characterization of molecular profiles within specific areas such as brain structures and biofluids is essential to unveil the molecular basis for structure specialization as well as the molecular imbalance associated with neurodegenerative and psychiatric diseases. AREAS COVERED: Much of our knowledge about brain functionality derives from neurophysiological, anatomical, and transcriptomic approaches. More recently, laser capture and imaging proteomics, technological and computational developments in LC-MS/MS, as well as antibody/aptamer-based platforms have allowed the generation of novel cellular, spatial, and posttranslational dimensions as well as innovative facets in biomarker validation and druggable target identification. EXPERT OPINION: Proteomics is a powerful toolbox to functionally characterize, quantify, and localize the extensive protein catalog of the human brain across physiological and pathological states. Brain function depends on multi-dimensional protein homeostasis, and its elucidation will help us to characterize biological pathways that are essential to properly maintain cognitive functions. In addition, comprehensive human brain pathological proteomes may be the basis in computational drug-repositioning methods as a strategy for unveiling potential new therapies in neurodegenerative and psychiatric disorders.

Fractalkine in Health and Disease.

CX3CL1 is one of the 50 up-to-date identified and characterized chemokines. While other chemokines are produced as small, secreted proteins, CX3CL1 (fractalkine) is synthetized as a transmembrane protein which also leads to a soluble form produced as a result of proteolytic cleavage. The membrane-bound protein and the soluble forms exhibit different biological functions. While the role of the fractalkine/CX3CR1 signaling axis was described in the nervous system and was also related to the migration of leukocytes to sites of inflammation, its actions are controversial in cancer progression and anti-tumor immunity. In the present review, we first describe the known biology of fractalkine concerning its action through its cognate receptor, but also its role in the activation of different integrins. The second part of this review is dedicated to its role in cancer where we discuss its role in anti-cancer or procarcinogenic activities.

Drug-Induced Reorganisation of Lipid Metabolism Limits the Therapeutic Efficacy of Ponatinib in Glioma Stem Cells.

The standard of care for glioblastoma (GBM) involves surgery followed by adjuvant radio- and chemotherapy, but often within months, patients relapse, and this has been linked to glioma stem cells (GSCs), self-renewing cells with increased therapy resistance. The identification of the epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR) as key players in gliomagenesis inspired the development of inhibitors targeting these tyrosine kinases (TKIs). However, results from clinical trials testing TKIs have been disappointing, and while the role of GSCs in conventional therapy resistance has been extensively studied, less is known about resistance of GSCs to TKIs. In this study, we have used compartmentalised proteomics to analyse the adaptive response of GSCs to ponatinib, a TKI with activity against PDGFR. The analysis of differentially expressed proteins revealed that GSCs respond to ponatinib by broadly rewiring lipid metabolism, involving fatty acid beta-oxidation, cholesterol synthesis, and sphingolipid degradation. Inhibiting each of these metabolic pathways overcame ponatinib adaptation of GSCs, but interrogation of patient data revealed sphingolipid degradation as the most relevant pathway in GBM. Our data highlight that targeting lipid metabolism, and particularly sphingolipid degradation in combinatorial therapies, could improve the outcome of TKI therapies using ponatinib in GBM.

Involvement of Glucosamine 6 Phosphate Isomerase 2 (GNPDA2) Overproduction in ß-Amyloid- and Tau P301L-Driven Pathomechanisms.

Alzheimer's disease (AD) is a neurodegenerative olfactory disorder affecting millions of people worldwide. Alterations in the hexosamine- or glucose-related pathways have been described through AD progression. Specifically, an alteration in glucosamine 6 phosphate isomerase 2 (GNPDA2) protein levels has been observed in olfactory areas of AD subjects. However, the biological role of GNPDA2 in neurodegeneration remains unknown. Using mass spectrometry, multiple GNPDA2 interactors were identified in human nasal epithelial cells (NECs) mainly involved in intraciliary transport. Moreover, GNPDA2 overexpression induced an increment in NEC proliferation rates, accompanied by transcriptomic alterations in Type II interferon signaling or cellular stress responses. In contrast, the presence of beta-amyloid or mutated Tau-P301L in GNPDA2-overexpressing NECs induced a slowdown in the proliferative capacity in parallel with a disruption in protein processing. The proteomic characterization of Tau-P301L transgenic zebrafish embryos demonstrated that GNPDA2 overexpression interfered with collagen biosynthesis and RNA/protein processing, without inducing additional changes in axonal outgrowth defects or neuronal cell death. In humans, a significant increase in serum GNPDA2 levels was observed across multiple neurological proteinopathies (AD, Lewy body dementia, progressive supranuclear palsy, mixed dementia and amyotrophic lateral sclerosis) (n = 215). These data shed new light on GNPDA2-dependent mechanisms associated with the neurodegenerative process beyond the hexosamine route.

Proteomic profiling of human plasma extracellular vesicles identifies PF4 and C1R as novel biomarker in sarcopenia.

BACKGROUND: Sarcopenia, the gradual and generalized loss of muscle mass and function with ageing, is one of the major health problems in older adults, given its high prevalence and substantial socioeconomic implications. Despite the extensive efforts to reach consensus on definition and diagnostic tests and cut-offs for sarcopenia, there is an urgent and unmet need for non-invasive, specific and sensitive biomarkers for the disease. Extracellular vesicles (EVs) are present in different biofluids including plasma, whose cargo reflects cellular physiology. This work analysed EV proteome in sarcopenia and robust patients in the search for differentially contained proteins that can be used to diagnose the disease. METHODS: Plasma small EVs (sEVs) from a total of 29 robust controls (aged 73.4 ± 5.6 years; 11 men and 18 women) and 49 sarcopenic patients (aged 82.3 ± 5.4 years; 15 men and 34 women) aged 65 years and older were isolated and their cargo was analysed by proteomics. Proteins whose concentration in sEVs was different between sarcopenic and robust patients were further validated using ELISA. The concentration of these candidates was correlated to the EWGSOP2 sarcopenia tests for low muscle strength and low physical performance, and receiver operating characteristic (ROC) curve analyses were carried out to evaluate their diagnostic power, sensitivity and specificity. RESULTS: Proteomic analysis identified 157 sEVs proteins in both sarcopenic and robust samples. Among them, 48 proteins had never been reported in the ExoCarta nor Vesiclepedia databases. Statistical analysis revealed eight proteins whose concentration was significantly different between groups: PF4 (log2 FC = 4.806), OIT3 (log2 FC = -1.161), MMRN1 (log2 FC = -1.982), MASP1 (log2 FC = -0.627), C1R (log2 FC = 1.830), SVEP1 (log2 FC = 1.295), VCAN (FC = 0.937) and SPTB (log2 FC = 1.243). Among them, platelet factor 4 (PF4) showed the lowest concentration while Complement C1r subcomponent (C1R) increased the most in sarcopenic patients, being these results confirmed by ELISA (P = 1.07E-09 and P = 0.001287, respectively). The concentrations of candidate proteins significantly correlated with EWGSOP2 tests currently used. ROC curve analysis showed an area under the curve of 0.8921 and 0.7476 for PF4 and C1R, respectively. Choosing the optimal for PF4, 80% sensitivity and 85.71% specificity was reached while the optimal cut-off value of C1R would allow sarcopenia diagnosis with 75% sensitivity and 66.67% specificity. CONCLUSIONS: Our results support the determination of EV PF4 and C1R as plasma diagnostic biomarkers in sarcopenia and open the door to investigate the role of the content of these vesicles in the pathogeny of the disease.

PD-1/LAG-3 co-signaling profiling uncovers CBL ubiquitin ligases as key immunotherapy targets.

Many cancer patients do not benefit from PD-L1/PD-1 blockade immunotherapies. PD-1 and LAG-3 co-upregulation in T-cells is one of the major mechanisms of resistance by establishing a highly dysfunctional state in T-cells. To identify shared features associated to PD-1/LAG-3 dysfunctionality in human cancers and T-cells, multiomic expression profiles were obtained for all TCGA cancers immune infiltrates. A PD-1/LAG-3 dysfunctional signature was found which regulated immune, metabolic, genetic, and epigenetic pathways, but especially a reinforced negative regulation of the TCR signalosome. These results were validated in T-cell lines with constitutively active PD-1, LAG-3 pathways and their combination. A differential analysis of the proteome of PD-1/LAG-3 T-cells showed a specific enrichment in ubiquitin ligases participating in E3 ubiquitination pathways. PD-1/LAG-3 co-blockade inhibited CBL-B expression, while the use of a bispecific drug in clinical development also repressed C-CBL expression, which reverted T-cell dysfunctionality in lung cancer patients resistant to PD-L1/PD-1 blockade. The combination of CBL-B-specific small molecule inhibitors with anti-PD-1/anti-LAG-3 immunotherapies demonstrated notable therapeutic efficacy in models of lung cancer refractory to immunotherapies, overcoming PD-1/LAG-3 mediated resistance.