Summary Neuronal development and function rely on precise sorting of membrane proteins to distinct neuronal domains, yet the underlying mechanisms remain incompletely understood. Here, we investigated the trafficking of two highly homologous NLGNs (NLGN1 and 2) which distinctly localize and function at excitatory and inhibitory synapses. By using spatiotemporal proteomics, genetic engineering and microscopy in CNS cultured neurons and organotypic slices, we dissected the itinerary of biosynthetic NLGNs, identifying co-cargoes and sorting routes to dendrites and the AIS. At the AIS, which contains only inhibitory synapses, both NLGN1 and NLGN2 are locally exocytosed. While NLGN2 is stably recruited to axo-axonic synapses by its extracellular domain and modulated by activity, NLGN1 is retrieved by endocytic mechanisms. We propose that biosynthetic NLGNs are co-sorted to the AIS PM where they detect pre-synaptic type. These findings may help to elucidate the role of AIS-localized NLGNs in shaping AIS structural and functional plasticity. Highlights Spatiotemporal proteomics elucidates interactome of biosynthetic NLGN1 in neurons NLGN1 and NLGN2 are targeted to the AIS by kinesin-1 NLGN2, but not NLGN1, is retained at axo-axonic synapses and modulated by activity NLGN stability at the AIS plasma membrane is determined by the ectodomain Graphical Abstract

Macrophages are the most abundant immune cells in the prostate tumor microenvironment and capable of killing tumor cells, but tumor intrinsic modulators of resistance to the innate immune system are unknown. To identify genes essential for macrophage-mediated killing, we performed a genome-wide co-culture CRISPR screen and identified Androgen Receptor (AR), PRKCD, and multiple components of the NF-κB pathway (IKBKB/IKBKG/CHUK) as tumor-intrinsic essential factors to allow for macrophage-mediated killing. Mechanistically, both AR and NF-κB directly drive expression of PRKCD within cancer cells, functionally implicating all hits within one molecular pathway. Importantly, androgen deprivation and AR-inhibition both rendered tumor cells resistant to macrophage-mediated killing, which positions tumor-intrinsic AR signaling as a bona fide immunomodulatory pathway. Proteomic analyses showed a selective downregulation of the oxidative phosphorylation pathway in PRKCD- and IKBKG-KO cells, suggesting impaired mitochondrial function, which was confirmed by electron microscopy analyses. Finally, phosphoproteomic analyses revealed that all hits perturbing macrophage-mediated tumor cell eradication, impaired ferroptosis signaling in the tumor cells, which was confirmed transcriptionally using samples from a neoadjuvant phase II clinical trial with the AR-inhibitor enzalutamide. These data reveal immune protection from macrophages as an adverse consequence of hormonal therapy in prostate cancer patients.

Activating PIK3CA mutations are among the most frequent oncogenic drivers in breast cancer, with E545K and H1047R mutants representing the most prevalent hotspot variants. Despite the development of potent PI3K inhibitors, clinical efficacy remains limited in some cases. This underscores the need to understand how specific oncogenic PIK3CA mutations reshape signaling networks and therapeutic responses. Here, we compared the E545K and H1047R mutant breast epithelial cells to delineate mutation-specific signaling programs, growth phenotypes, and responses to PI3Kα inhibition in the presence and absence of insulin, using integrated growth assays and quantitative proteomic and phosphoproteomic profiling. These analyses uncovered mutation-specific signaling architectures and inhibitor sensitivities. Both mutants exhibited basal MAPK activation, but showed divergent MAPK phosphorylation dynamics in distinct PIK3CA mutations, suggesting a pivotal role for MAPK signaling. Upon PI3Kα inhibition with alpelisib, insulin engaged bypass signaling that partially counteracted downstream suppression. MEK inhibition alone suppressed the growth of PIK3CA mutant cells, and dual targeting of PI3K and MAPK signaling produced greater growth suppression than either single agent alone under insulin-stimulated conditions. Collectively, these findings reveal mutation-specific adaptive signaling and support combined PI3Kα and MAPK pathway inhibition as a strategy to improve therapeutic efficacy in PIK3CA mutant breast cancer.

Abstract Proximity labeling methods (including BioID, TurboID, and ultraID), along with surface proteomics and microdomain mapping, enable proteome-wide identification of spatially proximal proteins via MS-based analysis. These workflows require specific enrichment of biotinylated proteins using affinity purification, yet enrichment specificity can often be compromised by nonspecifically bound proteins. As labeling strategies are increasingly applied to complex biological samples with low protein input or low biotin stoichiometry, accurately distinguishing true targets from the background becomes a major analytical challenge. Despite its critical impact on data quality and interpretation, the influence of the biotinylation level and protein input on enrichment performance remains poorly characterized, limiting the reliability of proximity labeling experiments. To address this, we established a quantitative benchmarking framework that systematically evaluates biotin enrichment under controlled conditions, including scenarios of low biotin stoichiometry. Using this setup, we show that enrichment specificity strongly depends on biotin stoichiometry: higher levels of biotinylation in samples yield high specificity, whereas low biotinylation increases nonspecific background. Reduced protein input further limits the recovery of true targets, yet maintains enrichment specificity, highlighting sensitivity constraints of enrichment-based workflows. We apply this framework to biotinylated extracellular vesicle (EV) cargo uptake in recipient cells using ultraID-CD63 labeling. Detection of the most abundant EV cargo proteins under low-biotinylation conditions indicates that current workflows approach the lower bounds of biotin enrichment sensitivity. Together, these standards provide a practical reference for evaluating and optimizing biotin enrichment workflows, supporting quantitative and reproducible proximity labeling in proteomics.

Resistance to BRAF/MAPK inhibitors is a significant challenge in melanoma treatment, driven by adaptive and acquired mechanisms allowing tumor cells to evade therapy. We explored early signaling responses to BRAF and MAPK inhibition in a BRAFV600E-sensitive melanoma cell line and a drug-resistant ARID1A-knockout (KO) derivative. ARID1A, frequently mutated in melanoma, is linked to resistance and immune evasion. Through an innovative systems biology approach integrating multi-omics datasets, we identified critical resistance mechanisms. We found that ARID1A-KO cells exhibited transcriptional rewiring, sustaining MAPK1/3 and JNK activity post-treatment, suppressing PRKD1 activation, increasing JUN activity, and disrupting PKC dynamics via elevated RTKs (e.g., EGFR, ROS1) and Ephrin receptor activity. ARID1A-KO also reduced HLA-related protein expression and enhanced extracellular matrix components, potentially limiting immune infiltration and immunotherapy efficacy. Our multi-omics analysis revealed PRKD1, JUN, and NCK1 as key resistance nodes, offering potential targets for therapeutic strategies to counter resistance in melanoma.

Motor neurons derived from induced human pluripotent stem cells offer a powerful model to study motor neuron diseases, such as amyotrophic lateral sclerosis. While widely used, our knowledge of the proteomic changes in these models is rather rudimentary. In this study, we conducted a comparative proteomic analysis of induced pluripotent stem cell-derived motor neurons carrying amyotrophic lateral sclerosis-associated mutations in C9ORF72, TARDBP, or FUS. This revealed both mutation-specific and shared proteomic signatures, unveiling common and divergent disease mechanisms. Using these new insights, we then evaluated the therapeutic potential of mesenchymal stem cell-derived extracellular vesicles. These experiments showed a functional effect of mesenchymal stem cell-derived extracellular vesicles in amyotrophic lateral sclerosis-FUS motor neurons in vitro and their ability to reverse proteomic changes more generally in motor neurons with different amyotrophic lateral sclerosis genetic backgrounds. These findings highlight key molecular pathways involved in amyotrophic lateral sclerosis at the protein level and support the potential of mesenchymal stem cell-derived extracellular vesicles as a versatile therapeutic approach.

Synthetic lethal interactions (SLIs) based on genomic alterations in cancer have been therapeutically explored. We investigated the SLI space as a function of differential RNA expression in cancer and normal tissue. Computational analyses of functional genomic and gene expression resources uncovered a cancer-specific SLI between the paralogs cytidine diphosphate diacylglycerol synthase 1 (CDS1) and CDS2. The essentiality of CDS2 for cell survival is observed for mesenchymal-like cancers, which have low or absent CDS1 expression and account for roughly half of all cancers. Mechanistically, the CDS1-2 SLI is accompanied by disruption of lipid homeostasis, including accumulation of cholesterol esters and triglycerides, and apoptosis. Genome-wide CRISPR-Cas9 knockout screens in CDS1-negative cancer cells identify no common escape mechanism of death caused by CDS2 ablation, indicating the robustness of the SLI. Synthetic lethality is driven by CDS2 dosage and depends on catalytic activity. Thus, CDS2 may serve as a pharmacologically tractable target in mesenchymal-like cancers.

Proper neuronal development and function rely on the polarized sorting of transmembrane proteins (TMPs) to their distinct somatodendritic or axonal domains. Most TMPs follow a conventional secretory pathway confined to the cell soma, where they are translated at the rough ER, exit through ER exit sites (ERES) to reach the Golgi apparatus (GA), prior to their delivery to the plasma membrane (PM). Intriguingly, we recently found that many mRNAs encoding TMPs are associated with the axonally-enriched ER-protein P180, which facilitates axonal ER-ribosome interaction and regulates local translation. Given the absence of the GA in the axon, it remains unknown how locally synthesized TMPs leave the ER and reach the axonal PM. Here, we visualized the translation of different axonal TMPs within the axon and followed their trafficking from the ER. We found that a fraction of neosynthesized cargos follow an unconventional route, exiting the axonal ER and reaching the PM independent of the GA. Notably, we identified axonal ERES components tightly associated with the axonal ER. Removal of these components prevents TMP exit from the axonal ER, while addition of external cues increases the number of axonal ERES and Golgi-bypassing cargos. Moreover, we identified the ER-translation regulator HDLBP and the NRZ-SEC22B vesicular tether complex associated with axonal ERES and involved in unconventional secretion. Local removal of axonal ERES components or depletion of these identified players causes defects in axon growth and bouton assembly. We propose a novel mechanism coupling local TMP translation and secretion to the PM at the axonal ER, which is essential for neuronal development. HIGHLIGHTS ### Competing Interest Statement The authors have declared no competing interest. European Research Council, https://ror.org/0472cxd90, ERC-StG 950617, ERC-StG 101163280 Target ALS, https://ror.org/03fsqvg68, NI-2024-NAI-S5

Isolation and analysis of GFPhi NMuMG cells expressing Fgfr2-GFP variants using FACS (includes gating strategy) and western blotting.

The presentation of peptides on HLA molecules is essential to CD8+ T cell responses. Here, we show that loss of uL14 significantly downregulates the expression of antigen processing and presentation (APP) components in melanoma cell lines. Peptides generated following knockdown show different characteristics, with altered peptide charge, and differences in anchor residue positions. These peptides also have lower predicted binding to the HLA alleles and a shorter predicted HLA-peptide complex half-life. These result in a functional difference in APP, and knockdown of uL14 causes a reduction in the ability of CD8+ T cells to recognize and kill melanoma cells in a co-culture assay. Together, our data suggest that loss of uL14 alters the peptide pool available for presentation and thus may act as an escape mechanism from tumor immune surveillance.

Proteins identified in Fgfr2-GFP immunoprecipitation samples using MS-based proteomics. The table shows all proteins identified using global proteomics on GFP-immunoprecipitation samples using NMuMG cells expressing GFP, Fgfr2FL-GFP, or Fgfr2DE18- GFP.

Intercellular communication between T cells and cancer cells plays a pivotal role in determining cancer cell survival or death. Yet, our understanding of this interaction remains incomplete. Methods to study heterotypic cell interactions are either limited to targeted studies relying on predefined set of proteins, or require cell separation, thus disrupting the native environment. Stable isotope labeling by amino acids in cell culture (SILAC) enables proteome distinction in heterologous co-cultures without the need for physical separation. But dynamic studies remain constrained by the need for numerous mass spectrometry (MS) runs, the challenges in detecting low-abundant proteins, particularly in immune cells and the limited data completeness due to the use of data-dependent MS1-based precursor quantification. To overcome these limitations, we evaluate the integration of SILAC with tandem mass tag (TMT) multiplexing and SILAC-directed real-time search (RTS). TMT labeling enables simultaneous analysis of multiple samples, while RTS-MS3 acquisition using SILAC-induced mass shifts as fixed modifications triggers MS3 scans for specific proteome populations within a mixed cell system, improving quantitative accuracy and proteome coverage for target protein populations. We benchmarked our acquisition methods using SILAC-labeled samples mixed at defined ratios and validated the approach in biologically relevant co-culture experiments. Additionally, we introduced a carrier channel to enhance detection of lower-abundant T cell proteins, while maintaining acceptable quantitative precision. Our results demonstrate that the combined SILAC-TMT-RTS strategy dramatically improves proteome depth, temporal resolution, and cell-type specificity for short-term co-culture interaction proteomics studies. In co-culture samples of T cells with non-small cell lung cancer cell lines that were either sensitive or resistant to T cell killing, our method revealed candidate mechanisms underlying their differential sensitivity. Our integrated approach combining SILAC, TMT and RTS to resolve cell-specific proteome dynamics in co-culture represents a novel and powerful advance. ### Competing Interest Statement The authors have declared no competing interest. * ACN : acetonitrile DDA : data-dependent acquisition FDR : false-discovery rate TMT : tandem mass tag LC-MS/MS : liquid chromatography coupled to tandem mass spectrometry MS : mass spectrometry NSCLC : non-small cell lung cancer PSM : peptide-spectrum match RTS : real-time search RTS-H : real-time search with heavy SILAC as a fixed modification RTS-I : real-time search with intermediate SILAC as a fixed modification RTS-L : real-time search with light SILAC as a fixed modification SILAC : stable isotope labeling by amino acids in cell culture S/N : signal-to-noise SPS : synchronous precursor selection TMT : tandem mass tags Netherlands Organization for Scientific Research, OCENW.XL21.XL21.027 National Road Map for Large-scale Infrastructures, 184.034.019

Key resources. List of antibodies, commercial assays, experimental models, reagents, and software used in this study.

Exact P values corresponding to Fig. 4B.

Enhancing the immunogenicity of tumor cells is a major objective in cancer therapy, particularly for tumors with low immune cell infiltration scores. Inducing immunogenic forms of programmed cell death (PCD) offers a promising strategy to strengthen anti-tumor immunity and improve therapeutic outcomes. Necroptosis, a highly inflammatory form of regulated cell death triggered by TNF signaling, can elicit robust immune activation. However, its regulation in tumor cells remains incompletely understood, limiting its therapeutic exploitation. To investigate the protein-protein interactions that govern necroptotic cell death in tumor cells, we established a co-Immunoprecipitation - Mass Spectrometry (coIP-MS) workflow using RIPK3, the central effector kinase driving necroptosis in TNF-induced signaling, as bait. This unbiased proteomic approach enables the identification of candidate regulators directly associated with the necrosome complex components under active necroptotic conditions. Among identified candidates, TCOF1 and GDF15 emerged as previously unrecognized modulators, with functional knockout of either gene markedly enhancing necroptotic cell death in tumor cells. Reciprocal IP experiments confirmed a direct interaction between GDF15 and RIPK3, supporting its mechanistic role as a negative regulator that suppresses necroptotic signaling. Thus, our findings extend the function of GDF15 beyond its established role in inflammation, uncovering an additional layer of regulation at the level of cell-intrinsic death signaling. Collectively, our findings position GDF15 as a RIPK3-interacting “brake” on necroptotic cell death and highlight TCOF1 as an additional inhibitory node. Our study underscores the potential of targeting necroptosis-suppressive mechanisms to influence PCD outcomes in tumors and demonstrates the power of coIP-MS for mapping TNF-induced interactions to reveal actionable molecular targets for tumor sensitization. ### Competing Interest Statement The authors have declared no competing interest. Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO), Project 184.034.019

Data supporting the in vitro and in vivo Fgfr2-E18 C-tail motif analyses.

BACKGROUND:Colorectal cancer (CRC) patients with inoperable peritoneal metastases (PM) have a dismal prognosis with limited treatment options. Local treatment of CRC-PM with oxaliplatin is commonly applied, but biomarkers steering patient selection, or informing potentially effective combination therapies are lacking. A novel potentially effective treatment strategy is Pressurized IntraPeritoneal Aerosol Chemotherapy (PIPAC) in which CRC-PM are exposed to cyclic treatment with high concentrations of locally applied oxaliplatin. However, it is unclear whether and how CRC-PM respond to PIPAC. METHODS:Here, we generated a biobank from 20 patients receiving PIPAC with oxaliplatin for CRC-PM. The biobank contains biopsies from 3 PM per patient, repeatedly sampled prior to each treatment cycle, and ascites. Anti-tumor effects were analyzed by shallow single-cell karyotype sequencing (sc-karyoSeq). RNA-sequencing and proteomics were performed to assess changes in gene and protein expression. Immunohistochemistry was performed to assess treatment-induced changes in tissue histology. Ascites was used to assess immunoglobulin content and reactivity. RESULTS:PIPAC reduced genomic heterogeneity and aneuploidy scores among PIPAC-surviving tumor cells. Furthermore, PIPAC reduced immunosuppressive signals (hypoxia, interleukin-10, transforming growth factor β), and induced an influx of B and T lymphocytes, which organized into metastasis-associated Tertiary Lymphoid Structures (TLS). TLS are biomarkers predicting response to Immune-Checkpoint Inhibitors (ICIs). The T cells residing in PIPAC-induced TLS expressed high levels of the checkpoints PD-1, TIGIT and EBI3. PIPAC also caused the generation of plasma cells producing tumor-reactive antibodies. CONCLUSION:PIPAC shows modest anti-tumor activity and induces immune parameters predicting response to ICIs. Patients with inoperable CRC-PM may therefore benefit from PIPAC in combination with ICIs.

Supplementary Methods related to FLAG and HALO tag constructs.

Intestinal stem cells (ISCs) face the challenge of integrating metabolic demands with unique regenerative functions. Studies have shown an intricate interplay between metabolism and stem cell capacity; however, it is still not understood how this process is regulated. Combining ribosome profiling and CRISPR screening in intestinal organoids, we identify the nascent polypeptide–associated complex (NAC) as a key mediator of this process. Our findings suggest that NAC is responsible for relocalizing ribosomes to the mitochondria and regulating ISC metabolism. Upon NAC inhibition, intestinal cells show decreased import of mitochondrial proteins, which are needed for oxidative phosphorylation, and, consequently, enable the cell to maintain a stem cell identity. Furthermore, we show that overexpression of NACα is sufficient to drive mitochondrial respiration and promote ISC identity. Ultimately, our results reveal the pivotal role of NAC in regulating ribosome localization, mitochondrial metabolism, and ISC function, providing insights into the potential mechanism behind it.

Alignments of C-tail sequences of Fgfr2 orthologs and human Fgfr paralogs.