Region-specific and age-related differences in astrocytes in the human brain.

Astrocyte heterogeneity and its relation to aging in the normal human brain remain poorly understood. We here analyzed astrocytes in gray and white matter brain tissues obtained from donors ranging in age between the neonatal period to over 100 years. We show that astrocytes are differently distributed with higher density in the white matter. This regional difference in cellular density becomes less prominent with age. Additionally, we confirm the presence of morphologically distinct astrocytes, with gray matter astrocytes being morphologically more complex. Notably, gray matter astrocytes morphologically change with age, while white matter astrocytes remain relatively consistent in morphology. Using regional mass spectrometry-based proteomics, we did, however, identify astrocyte specific proteins with regional differences in abundance, reflecting variation in cellular density or expression level. Importantly, the expression of some astrocyte specific proteins region-dependently decreases with age. Taken together, we provide insights into region- and age-related differences in astrocytes in the human brain.

Axonal endoplasmic reticulum tubules control local translation via P180/RRBP1-mediated ribosome interactions.

Local mRNA translation in axons is critical for the spatiotemporal regulation of the axonal proteome. A wide variety of mRNAs are localized and translated in axons; however, how protein synthesis is regulated at specific subcellular sites in axons remains unclear. Here, we establish that the axonal endoplasmic reticulum (ER) supports axonal translation in developing rat hippocampal cultured neurons. Axonal ER tubule disruption impairs local translation and ribosome distribution. Using nanoscale resolution imaging, we find that ribosomes make frequent contacts with axonal ER tubules in a translation-dependent manner and are influenced by specific extrinsic cues. We identify P180/RRBP1 as an axonally distributed ribosome receptor that regulates local translation and binds to mRNAs enriched for axonal membrane proteins. Importantly, the impairment of axonal ER-ribosome interactions causes defects in axon morphology. Our results establish a role for the axonal ER in dynamically localizing mRNA translation, which is important for proper neuron development.

Deciphering lineage specification during early embryogenesis in mouse gastruloids using multilayered proteomics.

Gastrulation is a critical stage in embryonic development during which the germ layers are established. Advances in sequencing technologies led to the identification of gene regulatory programs that control the emergence of the germ layers and their derivatives. However, proteome-based studies of early mammalian development are scarce. To overcome this, we utilized gastruloids and a multilayered mass spectrometry-based proteomics approach to investigate the global dynamics of (phospho) protein expression during gastruloid differentiation. Our findings revealed many proteins with temporal expression and unique expression profiles for each germ layer, which we also validated using single-cell proteomics technology. Additionally, we profiled enhancer interaction landscapes using P300 proximity labeling, which revealed numerous gastruloid-specific transcription factors and chromatin remodelers. Subsequent degron-based perturbations combined with single-cell RNA sequencing (scRNA-seq) identified a critical role for ZEB2 in mouse and human somitogenesis. Overall, this study provides a rich resource for developmental and synthetic biology communities endeavoring to understand mammalian embryogenesis.

Proteomic dissection of vanishing white matter pathogenesis.

Vanishing white matter (VWM) is a leukodystrophy caused by biallelic pathogenic variants in eukaryotic translation initiation factor 2B. To date, it remains unclear which factors contribute to VWM pathogenesis. Here, we investigated the basis of VWM pathogenesis using the 2b5ho mouse model. We first mapped the temporal proteome in the cerebellum, corpus callosum, cortex, and brainstem of 2b5ho and wild-type (WT) mice. Protein changes observed in 2b5ho mice were then cross-referenced with published proteomic datasets from VWM patient brain tissue to define alterations relevant to the human disease. By comparing 2b5ho mice with their region- and age-matched WT counterparts, we showed that the proteome in the cerebellum and cortex of 2b5ho mice was already dysregulated prior to pathology development, whereas proteome changes in the corpus callosum only occurred after pathology onset. Remarkably, protein changes in the brainstem were transient, indicating that a compensatory mechanism might occur in this region. Importantly, 2b5ho mouse brain proteome changes reflect features well-known in VWM. Comparison of the 2b5ho mouse and VWM patient brain proteomes revealed shared changes. These could represent changes that contribute to the disease or even drive its progression in patients. Taken together, we show that the 2b5ho mouse brain proteome is affected in a region- and time-dependent manner. We found that the 2b5ho mouse model partly replicates the human disease at the protein level, providing a resource to study aspects of VWM pathogenesis by highlighting alterations from early to late disease stages, and those that possibly drive disease progression.

Predicting treatment outcome using kinome activity profiling in HER2+ breast cancer biopsies.

In this study, we measured the kinase activity profiles of 32 pre-treatment tumor biopsies of HER2-positive breast cancer patients. The aim of this study was to assess the prognostic potential of kinase activity levels, to identify potential mechanisms of resistance and to predict treatment success of HER2-targeted therapy combined with chemotherapy. Indeed, our system-wide kinase activity analysis allowed us to link kinase activity to treatment response. Overall, high kinase activity in the HER2-pathway was associated with good treatment outcome. We found eleven kinases differentially regulated between treatment outcome groups, including well-known players in therapy resistance, such as p38a, ERK, and FAK, and an unreported one, namely MARK1. Lastly, we defined an optimal signature of four kinases in a multiple logistic regression diagnostic test for prediction of treatment outcome (AUC = 0.926). This kinase signature showed high sensitivity and specificity, indicating its potential as predictive biomarker for treatment success of HER2-targeted therapy.

Multimodal stimulation screens reveal unique and shared genes limiting T cell fitness.

Genes limiting T cell antitumor activity may serve as therapeutic targets. It has not been systematically studied whether there are regulators that uniquely or broadly contribute to T cell fitness. We perform genome-scale CRISPR-Cas9 knockout screens in primary CD8 T cells to uncover genes negatively impacting fitness upon three modes of stimulation: (1) intense, triggering activation-induced cell death (AICD); (2) acute, triggering expansion; (3) chronic, causing dysfunction. Besides established regulators, we uncover genes controlling T cell fitness either specifically or commonly upon differential stimulation. Dap5 ablation, ranking highly in all three screens, increases translation while enhancing tumor killing. Loss of Icam1-mediated homotypic T cell clustering amplifies cell expansion and effector functions after both acute and intense stimulation. Lastly, Ctbp1 inactivation induces functional T cell persistence exclusively upon chronic stimulation. Our results functionally annotate fitness regulators based on their unique or shared contribution to traits limiting T cell antitumor activity.

Assessment of Kinome-Wide Activity Remodeling upon Picornavirus Infection.

Picornaviridae represent a large family of single-stranded positive RNA viruses of which different members can infect both humans and animals. These include the enteroviruses (e.g., poliovirus, coxsackievirus, and rhinoviruses) as well as the cardioviruses (e.g., encephalomyocarditis virus). Picornaviruses have evolved to interact with, use, and/or evade cellular host systems to create the optimal environment for replication and spreading. It is known that viruses modify kinase activity during infection, but a proteome-wide overview of the (de)regulation of cellular kinases during picornavirus infection is lacking. To study the kinase activity landscape during picornavirus infection, we here applied dedicated targeted mass spectrometry-based assays covering ∼40% of the human kinome. Our data show that upon infection, kinases of the MAPK pathways become activated (e.g., ERK1/2, RSK1/2, JNK1/2/3, and p38), while kinases involved in regulating the cell cycle (e.g., CDK1/2, GWL, and DYRK3) become inactivated. Additionally, we observed the activation of CHK2, an important kinase involved in the DNA damage response. Using pharmacological kinase inhibitors, we demonstrate that several of these activated kinases are essential for the replication of encephalomyocarditis virus. Altogether, the data provide a quantitative understanding of the regulation of kinome activity induced by picornavirus infection, providing a resource important for developing novel antiviral therapeutic interventions.

Innovative strategies for measuring kinase activity to accelerate the next wave of novel kinase inhibitors.

The development of protein kinase inhibitors (PKIs) has gained significance owing to their therapeutic potential for diseases like cancer. In addition, there has been a rise in refining kinase activity assays, each possessing unique biological and analytical characteristics crucial for PKI development. However, the PKI development pipeline experiences high attrition rates and approved PKIs exhibit unexploited potential because of variable patient responses. Enhancing PKI development efficiency involves addressing challenges related to understanding the PKI mechanism of action and employing biomarkers for precision medicine. Selecting appropriate kinase activity assays for these challenges can overcome these attrition rate issues. This review delves into the current obstacles in kinase inhibitor development and elucidates kinase activity assays that can provide solutions.

CAMSAPs and nucleation-promoting factors control microtubule release from γ-TuRC.

γ-Tubulin ring complex (γ-TuRC) is the major microtubule-nucleating factor. After nucleation, microtubules can be released from γ-TuRC and stabilized by other proteins, such as CAMSAPs, but the biochemical cross-talk between minus-end regulation pathways is poorly understood. Here we reconstituted this process in vitro using purified components. We found that all CAMSAPs could bind to the minus ends of γ-TuRC-attached microtubules. CAMSAP2 and CAMSAP3, which decorate and stabilize growing minus ends but not the minus-end tracking protein CAMSAP1, induced microtubule release from γ-TuRC. CDK5RAP2, a γ-TuRC-interactor, and CLASP2, a regulator of microtubule growth, strongly stimulated γ-TuRC-dependent microtubule nucleation, but only CDK5RAP2 suppressed CAMSAP binding to γ-TuRC-anchored minus ends and their release. CDK5RAP2 also improved selectivity of γ-tubulin-containing complexes for 13- rather than 14-protofilament microtubules in microtubule-capping assays. Knockout and overexpression experiments in cells showed that CDK5RAP2 inhibits the formation of CAMSAP2-bound microtubules detached from the microtubule-organizing centre. We conclude that CAMSAPs can release newly nucleated microtubules from γ-TuRC, whereas nucleation-promoting factors can differentially regulate this process.

Proteomics on malignant pleural effusions reveals ERα loss in metastatic breast cancer associates with SGK1-NDRG1 deregulation.

Breast cancer (BCa) is a highly heterogeneous disease, with hormone receptor status being a key factor in patient prognostication and treatment decision-making. The majority of primary tumours are positive for oestrogen receptor alpha (ERα), which plays a key role in tumorigenesis and disease progression, and represents the major target for treatment of BCa. However, around one-third of patients with ERα-positive BCa relapse and progress into the metastatic stage, with 20% of metastatic cases characterised by loss of ERα expression after endocrine treatment, known as ERα-conversion. It remains unclear whether ERα-converted cancers are biologically similar to bona fide ERα-negative disease and which signalling cascades compensate for ERα loss and drive tumour progression. To better understand the biological changes that occur in metastatic BCa upon ERα loss, we performed (phospho)proteomics analysis of 47 malignant pleural effusions derived from 37 BCa patients, comparing ERα-positive, ERα-converted and ERα-negative cases. Our data revealed that the loss of ERα-dependency in this metastatic site leads to only a partial switch to an ERα-negative molecular phenotype, with preservation of a luminal-like proteomic landscape. Furthermore, we found evidence for decreased activity of several key kinases, including serum/glucocorticoid regulated kinase 1 (SGK1), in ERα-converted metastases. Loss of SGK1 substrate phosphorylation may compensate for the loss of ERα-dependency in advanced disease and exposes a potential therapeutic vulnerability that may be exploited in treating these patients.

Phosphoprotein dynamics of interacting T cells and tumor cells by HySic.

Functional interactions between cytotoxic T cells and tumor cells are central to anti-cancer immunity. However, our understanding of the proteins involved is limited. Here, we present HySic (hybrid quantification of stable isotope labeling by amino acids in cell culture [SILAC]-labeled interacting cells) as a method to quantify protein and phosphorylation dynamics between and within physically interacting cells. Using co-cultured T cells and tumor cells, we directly measure the proteome and phosphoproteome of engaged cells without the need for physical separation. We identify proteins whose abundance or activation status changes upon T cell:tumor cell interaction and validate our method with established signal transduction pathways including interferon γ (IFNγ) and tumor necrosis factor (TNF). Furthermore, we identify the RHO/RAC/PAK1 signaling pathway to be activated upon cell engagement and show that pharmacologic inhibition of PAK1 sensitizes tumor cells to T cell killing. Thus, HySic is a simple method to study rapid protein signaling dynamics in physically interacting cells that is easily extended to other biological systems.

Luminal breast cancer identity is determined by loss of glucocorticoid receptor activity.

Glucocorticoid receptor (GR) is a transcription factor that plays a crucial role in cancer biology. In this study, we utilized an in silico-designed GR activity signature to demonstrate that GR relates to the proliferative capacity of numerous primary cancer types. In breast cancer, the GR activity status determines luminal subtype identity and has implications for patient outcomes. We reveal that GR engages with estrogen receptor (ER), leading to redistribution of ER on the chromatin. Notably, GR activation leads to upregulation of the ZBTB16 gene, encoding for a transcriptional repressor, which controls growth in ER-positive breast cancer and associates with prognosis in luminal A patients. In relation to ZBTB16's repressive nature, GR activation leads to epigenetic remodeling and loss of histone acetylation at sites proximal to cancer-driving genes. Based on these findings, epigenetic inhibitors reduce viability of ER-positive breast cancer cells that display absence of GR activity. Our findings provide insights into how GR controls ER-positive breast cancer growth and may have implications for patients' prognostication and provide novel therapeutic candidates for breast cancer treatment.

Deep (phospho)proteomics profiling of pre- treatment needle biopsies identifies signatures of treatment resistance in HER2+ breast cancer.

Patients with early-stage HER2-overexpressing breast cancer struggle with treatment resistance in 20%-40% of cases. More information is needed to predict HER2 therapy response and resistance in vivo. In this study, we perform (phospho)proteomics analysis of pre-treatment HER2+ needle biopsies of early-stage invasive breast cancer to identify molecular signatures predictive of treatment response to trastuzumab, pertuzumab, and chemotherapy. Our data show that accurate quantification of the estrogen receptor (ER) and HER2 biomarkers, combined with the assessment of associated biological features, has the potential to enable better treatment outcome prediction. In addition, we identify cellular mechanisms that potentially precondition tumors to resist therapy. We find proteins with expression changes that correlate with resistance and constitute to a strong predictive signature for treatment success in our patient cohort. Our results highlight the multifactorial nature of drug resistance in vivo and demonstrate the necessity of deep tumor profiling.

A cyclin-dependent kinase-mediated phosphorylation switch of disordered protein condensation.

Cell cycle transitions result from global changes in protein phosphorylation states triggered by cyclin-dependent kinases (CDKs). To understand how this complexity produces an ordered and rapid cellular reorganisation, we generated a high-resolution map of changing phosphosites throughout unperturbed early cell cycles in single Xenopus embryos, derived the emergent principles through systems biology analysis, and tested them by biophysical modelling and biochemical experiments. We found that most dynamic phosphosites share two key characteristics: they occur on highly disordered proteins that localise to membraneless organelles, and are CDK targets. Furthermore, CDK-mediated multisite phosphorylation can switch homotypic interactions of such proteins between favourable and inhibitory modes for biomolecular condensate formation. These results provide insight into the molecular mechanisms and kinetics of mitotic cellular reorganisation.

MYC is a clinically significant driver of mTOR inhibitor resistance in breast cancer.

Targeting the PI3K-AKT-mTOR pathway is a promising therapeutic strategy for breast cancer treatment. However, low response rates and development of resistance to PI3K-AKT-mTOR inhibitors remain major clinical challenges. Here, we show that MYC activation drives resistance to mTOR inhibitors (mTORi) in breast cancer. Multiomic profiling of mouse invasive lobular carcinoma (ILC) tumors revealed recurrent Myc amplifications in tumors that acquired resistance to the mTORi AZD8055. MYC activation was associated with biological processes linked to mTORi response and counteracted mTORi-induced translation inhibition by promoting translation of ribosomal proteins. In vitro and in vivo induction of MYC conferred mTORi resistance in mouse and human breast cancer models. Conversely, AZD8055-resistant ILC cells depended on MYC, as demonstrated by the synergistic effects of mTORi and MYCi combination treatment. Notably, MYC status was significantly associated with poor response to everolimus therapy in metastatic breast cancer patients. Thus, MYC is a clinically relevant driver of mTORi resistance that may stratify breast cancer patients for mTOR-targeted therapies.

A genome-wide CRISPR screen in human prostate cancer cells reveals drivers of macrophage-mediated cell killing and positions AR as a tumor-intrinsic immunomodulator.

The crosstalk between prostate cancer (PCa) cells and the tumor microenvironment plays a pivotal role in disease progression and metastasis and could provide novel opportunities for patient treatment. Macrophages are the most abundant immune cells in the prostate tumor microenvironment (TME) and are capable of killing tumor cells. To identify genes in the tumor cells that are critical for macrophage-mediated killing, we performed a genome-wide co-culture CRISPR screen and identified AR, PRKCD, and multiple components of the NF-κB pathway as hits, whose expression in the tumor cell are essential for being targeted and killed by macrophages. These data position AR signaling as an immunomodulator, and confirmed by androgen-deprivation experiments, that rendered hormone-deprived tumor cells resistant to macrophage-mediated killing. Proteomic analyses showed a downregulation of oxidative phosphorylation in the PRKCD- and IKBKG-KO cells compared to the control, suggesting impaired mitochondrial function, which was confirmed by electron microscopy analyses. Furthermore, phosphoproteomic analyses revealed that all hits impaired ferroptosis signaling, which was validated transcriptionally using samples from a neoadjuvant clinical trial with the AR-inhibitor enzalutamide. Collectively, our data demonstrate that AR functions together with the PRKCD and the NF-κB pathway to evade macrophage-mediated killing. As hormonal intervention represents the mainstay therapy for treatment of prostate cancer patients, our findings may have direct implications and provide a plausible explanation for the clinically observed persistence of tumor cells despite androgen deprivation therapy.

Optimized Suspension Trapping Method for Phosphoproteomics Sample Preparation.

A successful mass spectrometry-based phosphoproteomics analysis relies on effective sample preparation strategies. Suspension trapping (S-Trap) is a novel, rapid, and universal method of sample preparation that is increasingly applied in bottom-up proteomics studies. However, the performance of the S-Trap protocol for phosphoproteomics studies is unclear. In the existing S-Trap protocol, the addition of phosphoric acid (PA) and methanol buffer creates a fine protein suspension to capture proteins on a filter and is a critical step for subsequent protein digestion. Herein, we demonstrate that this addition of PA is detrimental to downstream phosphopeptide enrichment, rendering the standard S-Trap protocol suboptimal for phosphoproteomics. In this study, the performance of the S-Trap digestion for proteomics and phosphoproteomics is systematically evaluated in large-scale and small-scale samples. The results of this comparative analysis show that an optimized S-Trap approach, where trifluoroacetic acid is substituted for PA, is a simple and effective method to prepare samples for phosphoproteomics. Our optimized S-Trap protocol is applied to extracellular vesicles to demonstrate superior sample preparation workflow for low-abundance, membrane-rich samples.

Elucidating Fibroblast Growth Factor-Induced Kinome Dynamics Using Targeted Mass Spectrometry and Dynamic Modeling.

Fibroblast growth factors (FGFs) are paracrine or endocrine signaling proteins that, activated by their ligands, elicit a wide range of health and disease-related processes, such as cell proliferation and the epithelial-to-mesenchymal transition. The detailed molecular pathway dynamics that coordinate these responses have remained to be determined. To elucidate these, we stimulated MCF-7 breast cancer cells with either FGF2, FGF3, FGF4, FGF10, or FGF19. Following activation of the receptor, we quantified the kinase activity dynamics of 44 kinases using a targeted mass spectrometry assay. Our system-wide kinase activity data, supplemented with (phospho)proteomics data, reveal ligand-dependent distinct pathway dynamics, elucidate the involvement of not earlier reported kinases such as MARK, and revise some of the pathway effects on biological outcomes. In addition, logic-based dynamic modeling of the kinome dynamics further verifies the biological goodness-of-fit of the predicted models and reveals BRAF-driven activation upon FGF2 treatment and ARAF-driven activation upon FGF4 treatment.

Regional vulnerability of brain white matter in vanishing white matter.

Vanishing white matter (VWM) is a leukodystrophy that primarily manifests in young children. In this disease, the brain white matter is differentially affected in a predictable pattern with telencephalic brain areas being most severely affected, while others remain allegedly completely spared. Using high-resolution mass spectrometry-based proteomics, we investigated the proteome patterns of the white matter in the severely affected frontal lobe and normal appearing pons in VWM and control cases to identify molecular bases underlying regional vulnerability. By comparing VWM patients to controls, we identified disease-specific proteome patterns. We showed substantial changes in both the VWM frontal and pons white matter at the protein level. Side-by-side comparison of brain region-specific proteome patterns further revealed regional differences. We found that different cell types were affected in the VWM frontal white matter than in the pons. Gene ontology and pathway analyses identified involvement of region specific biological processes, of which pathways involved in cellular respiratory metabolism were overarching features. In the VWM frontal white matter, proteins involved in glycolysis/gluconeogenesis and metabolism of various amino acids were decreased compared to controls. By contrast, in the VWM pons white matter, we found a decrease in proteins involved in oxidative phosphorylation. Taken together, our data show that brain regions are affected in parallel in VWM, but to different degrees. We found region-specific involvement of different cell types and discovered that cellular respiratory metabolism is likely to be differentially affected across white matter regions in VWM. These region-specific changes help explain regional vulnerability to pathology in VWM.

Natural deletion of mouse carboxylesterases Ces1c/d/e impacts drug metabolism and metabolic syndrome development.

Mammalian carboxylesterase 1 enzymes can hydrolyze many xenobiotic chemicals and endogenous lipids. We here identified and characterized a mouse strain (FVB/NKI) in which three of the eight Ces1 genes were spontaneously deleted, removing Ces1c and Ces1e partly, and Ces1d entirely. We studied the impact of this Ces1c/d/e deficiency on drug and lipid metabolism and homeostasis. Ces1c/d/e-/- mice showed strongly impaired conversion of the anticancer prodrug irinotecan to its active metabolite SN-38 in plasma, spleen and lung. Plasma hydrolysis of the oral anticancer prodrug capecitabine to 5-DFCR was also profoundly reduced in Ces1c/d/e-/- mice. Our findings resolved previously unexplained FVB/NKI pharmacokinetic anomalies. On a medium-fat diet, Ces1c/d/e-/- female mice exhibited moderately higher body weight, mild inflammation in gonadal white adipose tissue (gWAT), and increased lipid load in brown adipose tissue (BAT). Ces1c/d/e-/- males showed more pronounced inflammation in gWAT and an increased lipid load in BAT. On a 5-week high-fat diet exposure, Ces1c/d/e deficiency predisposed to developing obesity, enlarged and fatty liver, glucose intolerance and insulin resistance, with severe inflammation in gWAT and increased lipid load in BAT. Hepatic proteomics analysis revealed that the acute phase response, involved in the dynamic cycle of immunometabolism, was activated in these Ces1c/d/e-/- mice. This may contribute to the obesity-related chronic inflammation and adverse metabolic disease in this strain. While Ces1c/d/e deficiency clearly exacerbated metabolic syndrome development, long-term (18-week) high-fat diet exposure overwhelmed many, albeit not all, observed phenotypic differences.