bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2025–03–09
twenty papers selected by
Ralitsa Radostinova Madsen, MRC-PPU
  1. Large-scale control over collective cell migration using light-activated epidermal growth factor receptors.
  2. Regulation of VEGFR3 signaling in lymphatic endothelial cells.
  3. Reproducible image-based profiling with Pycytominer.
  4. Visual Quality Control With CytoMDS, a Bioconductor Package for Low Dimensional Representation of Cytometry Sample Distances.
  5. Entering the era of deep single-cell proteomics.
  6. Packaged delivery of CRISPR-Cas9 ribonucleoproteins accelerates genome editing.
  7. Synergistic RAS-MAPK and AKT Activation in MYC-Driven Tumors via Adjacent PVT1 Rearrangements.
  8. A metabolic synthetic lethality of phosphoinositide 3-kinase-driven cancer.
  9. Precise Generation of Human Induced Pluripotent Stem Cell-derived Cell Lines Harboring Disease-relevant Single Nucleotide Variants Using a Prime Editing System.
  10. PerturbView: scalable image-based perturbation screens in cells and tissues.
  11. Sex Differences in VEGF and PDGF Ligand and Receptor Protein Expression during Adipose Tissue Expansion and Regression.
  12. Genetically encoded tension heterogeneity sculpts cardiac trabeculation.
  13. Unraveling cell-cell communication with NicheNet by inferring active ligands from transcriptomics data.
  14. FAK inhibition combined with the RAF-MEK clamp avutometinib overcomes resistance to targeted and immune therapies in BRAF V600E melanoma.
  15. Guidelines for minimal reporting requirements, design and interpretation of experiments involving the use of eukaryotic dual gene expression reporters (MINDR).
  16. A cellular and molecular basis of leptin resistance.
  17. TSC2 loss in neural progenitor cells suppresses mRNA translation of neurodevelopmental genes.
  18. Clonal memory of colitis accumulates and promotes tumor growth.
  19. Affinity Purification Mass Spectrometry on the Orbitrap-Astral Mass Spectrometer Enables High-Throughput Protein-Protein Interaction Mapping.
  20. Considerations for antibody-based detection of NRF2 in human cells.
  1. Cell Syst. 2025 Feb 26. pii: S2405-4712(25)00036-5. [Epub ahead of print] 101203
    Large-scale control over collective cell migration using light-activated epidermal growth factor receptors.
    Kevin Suh, Richard H Thornton, Long Nguyen, Payam E Farahani, Daniel J Cohen, Jared E Toettcher.
      Receptor tyrosine kinases (RTKs) play key roles in coordinating cell movement at both single-cell and tissue scales. The recent development of optogenetic tools for controlling RTKs and their downstream signaling pathways suggests that these responses may be amenable to engineering-based control for sculpting tissue shape and function. Here, we report that a light-controlled epidermal growth factor (EGF) receptor (OptoEGFR) can be deployed in epithelial cells for precise, programmable control of long-range tissue movements. We show that in OptoEGFR-expressing tissues, light can drive millimeter-scale cell rearrangements to densify interior regions or produce rapid outgrowth at tissue edges. Light-controlled tissue movements are driven primarily by phosphoinositide 3-kinase (PI3K) signaling, rather than diffusible ligands, tissue contractility, or ERK kinase signaling as seen in other RTK-driven migration contexts. Our study suggests that synthetic, light-controlled RTKs could serve as a powerful platform for controlling cell positions and densities for diverse applications, including wound healing and tissue morphogenesis.
    Keywords:  collective cell migration; epidermal growth factor receptor; optogenetics; tissue mechanics
    DOI:  https://doi.org/10.1016/j.cels.2025.101203
  2. Front Cell Dev Biol. 2025 ;13 1527971
    Regulation of VEGFR3 signaling in lymphatic endothelial cells.
    Kevin G Kuonqui, Adana-Christine Campbell, Bracha L Pollack, Jinyeon Shin, Ananta Sarker, Stav Brown, Hyeung Ju Park, Babak J Mehrara, Raghu P Kataru.
      The receptor tyrosine kinase vascular endothelial growth factor (VEGF) receptor 3 (VEGFR3) is the principal transmembrane receptor responsible for sensing and coordinating cellular responses to environmental lymphangiogenic stimuli in lymphatic endothelial cells (LECs). VEGFC and D (VEGFC/D) function as the cognate ligands to VEGFR3 by stimulating autophosphorylation of intracellular VEGFR3 tyrosine kinase domains that activate signal cascades involved in lymphatic growth and survival. VEGFR3 primarily promotes downstream signaling through the phosphoinositide 3-kinase (PI3K) and Ras signaling cascades that promote functions including cell proliferation and migration. The importance of VEGFR3 cascades in lymphatic physiology is underscored by identification of dysfunctional VEGFR3 signaling across several lymphatic-related diseases. Recently, our group has shown that intracellular modification of VEGFR3 signaling is a potent means of inducing lymphangiogenesis independent of VEGFC. This is important because long-term treatment with recombinant VEGFC may have deleterious consequences due to off-target effects. A more complete understanding of VEGFR3 signaling pathways may lead to novel drug development strategies. The purpose of this review is to 1) characterize molecular mediators of VEGFC/VEGFR3 downstream signaling activation and their functional roles in LEC physiology and 2) explore molecular regulation of overall VEGFR3 expression and activity within LECs.
    Keywords:  LECs; VEGFC; VEGFR3; lymphangiogenesis; lymphatics; lymphedema
    DOI:  https://doi.org/10.3389/fcell.2025.1527971
  3. Nat Methods. 2025 Mar 03.
    Reproducible image-based profiling with Pycytominer.
    Erik Serrano, Srinivas Niranj Chandrasekaran, Dave Bunten, Kenneth I Brewer, Jenna Tomkinson, Roshan Kern, Michael Bornholdt, Stephen J Fleming, Ruifan Pei, John Arevalo, Hillary Tsang, Vincent Rubinetti, Callum Tromans-Coia, Tim Becker, Erin Weisbart, Charlotte Bunne, Alexandr A Kalinin, Rebecca Senft, Stephen J Taylor, Nasim Jamali, Adeniyi Adeboye, Hamdah Shafqat Abbasi, Allen Goodman, Juan C Caicedo, Anne E Carpenter, Beth A Cimini, Shantanu Singh, Gregory P Way.
      Advances in high-throughput microscopy have enabled the rapid acquisition of large numbers of high-content microscopy images. Next, whether by deep learning or classical algorithms, image analysis pipelines commonly produce single-cell features. To process these single cells for downstream applications, we present Pycytominer, a user-friendly, open-source Python package that implements the bioinformatics steps key to image-based profiling. We demonstrate Pycytominer's usefulness in a machine-learning project to predict nuisance compounds that cause undesirable cell injuries.
    DOI:  https://doi.org/10.1038/s41592-025-02611-8
  4. Cytometry A. 2025 Mar 04.
    Visual Quality Control With CytoMDS, a Bioconductor Package for Low Dimensional Representation of Cytometry Sample Distances.
    Philippe Hauchamps, Simon Delandre, Stéphane T Temmerman, Dan Lin, Laurent Gatto.
      Quality Control (QC) of samples is an essential preliminary step in cytometry data analysis. Notably, the identification of potential batch effects and outlying samples is paramount to avoid mistaking these effects for true biological signals in downstream analyses. However, this task can prove to be delicate and tedious, especially for datasets with dozens of samples. Here, we present CytoMDS, a Bioconductor package implementing a dedicated method for low-dimensional representation of cytometry samples composed of marker expressions for up to millions of single cells. This method allows a global representation of all samples of a study, with one single point per sample, in such a way that projected distances can be visually interpreted. CytoMDS uses Earth Mover's Distance for assessing dissimilarities between multi-dimensional distributions of marker expression and Multi-Dimensional Scaling for low-dimensional projection of distances. Some additional visualization tools, both for projection quality diagnosis and for user interpretation of the projection coordinates, are also provided in the package. We demonstrate the strengths and advantages of CytoMDS for QC of cytometry data on three real biological datasets, revealing the presence of low-quality samples, batch effects, and biological signal between sample groups.
    DOI:  https://doi.org/10.1002/cyto.a.24921
  5. Nat Methods. 2025 Mar 03.
    Entering the era of deep single-cell proteomics.
    Ludwig R Sinn, Vadim Demichev.
      
    DOI:  https://doi.org/10.1038/s41592-025-02620-7
  6. Nucleic Acids Res. 2025 Feb 27. pii: gkaf105. [Epub ahead of print]53(5):
    Packaged delivery of CRISPR-Cas9 ribonucleoproteins accelerates genome editing.
    Hannah Karp, Madeline Zoltek, Kevin Wasko, Angel Luis Vazquez, Jinna Brim, Wayne Ngo, Alanna Schepartz, Jennifer A Doudna.
      Effective genome editing requires a sufficient dose of CRISPR-Cas9 ribonucleoproteins (RNPs) to enter the target cell while minimizing immune responses, off-target editing, and cytotoxicity. Clinical use of Cas9 RNPs currently entails electroporation into cells ex vivo, but no systematic comparison of this method to packaged RNP delivery has been made. Here we compared two delivery strategies, electroporation and enveloped delivery vehicles (EDVs), to investigate the Cas9 dosage requirements for genome editing. Using fluorescence correlation spectroscopy, we determined that >1300 Cas9 RNPs per nucleus are typically required for productive genome editing. EDV-mediated editing was >30-fold more efficient than electroporation, and editing occurs at least 2-fold faster for EDV delivery at comparable total Cas9 RNP doses. We hypothesize that differences in efficacy between these methods result in part from the increased duration of RNP nuclear residence resulting from EDV delivery. Our results directly compare RNP delivery strategies, showing that packaged delivery could dramatically reduce the amount of CRISPR-Cas9 RNPs required for experimental or clinical genome editing.
    DOI:  https://doi.org/10.1093/nar/gkaf105
  7. bioRxiv. 2025 Feb 22. pii: 2025.02.17.638454. [Epub ahead of print]
    Synergistic RAS-MAPK and AKT Activation in MYC-Driven Tumors via Adjacent PVT1 Rearrangements.
    Ashutosh Tiwari, Utkarsha Paithane, Jordan Friedlein, Kojiro Tashiro, Olivier Saulnier, Karina Barbosa, Quang Trinh, Bryan Hall, Shrawantee Saha, Aditi Soni, Takuma Nakashima, Andrey Bobkov, Lynn Miya Fujimoto, Rabi Murad, Svetlana Maurya, Mayank Saraswat, Shahab Sarmashghi, Joshua T Lange, Sihan Wu, Meher Beigi Masihi, Srija Ghosh, Gazal Hemmati, Owen Chapman, Liam Hendrikse, Brian James, Jens Luebeck, Tanja Eisemann, Theophilos Tzaridis, Deepak Rohila, Robyn Leary, Jyotika Varshney, Badrinath Konety, Scott M Dehm, Yasuhiko Kawakami, Rameen Beroukhim, David A Largaespada, Lincoln Stein, Lukas Chavez, Hiromichi Suzuki, William A Weiss, Jianhua Zhao, Aniruddha Deshpande, Robert J Wechsler-Reya, Michael D Taylor, Anindya Bagchi.
      MYC-driven (MYC+) cancers are aggressive and often fatal. MYC dysregulation is a key event in these cancers, but overexpression of MYC alone is not always enough to cause cancer. Plasmocytoma Variant Translocation 1 ( PVT1 ), a long non-coding RNA (lncRNA) adjacent to MYC on chromosome 8 is a rearrangement hotspot in many MYC+ cancers. In addition to being co-amplified with MYC, the genomic rearrangement at PVT1 involves translocation, which has had obscure functional consequences. We report that translocation at the PVT1 locus cause asymmetric enrichment of 5'-PVT1 and loss of 3'-PVT1. Despite being classified as a non-coding RNA, the retained 5' region of PVT1 generates a circular RNA (CircPVT1) that codes for the novel peptide we call Firefox (FFX). FFX augments AKT signaling and synergistically activates MYC and mTORC1 in these cells. Further, the 3' end of PVT1, which is lost during the translocation, codes for a tumor-suppressing micropeptide we named as Honeybadger (HNB). We demonstrate that HNB interacts with KRAS and disrupts the activation of KRAS effectors. Loss of HNB leads to activation of RAS/MAPK signaling pathway, and enhances MYC stability by promoting phosphorylation of MYC at Ser 62 . These findings identify PVT1 as a critical node that synchronizes MYC, AKT, and RAS-MAPK activities in cancer. Our study thus identifies a key mechanism by which rearrangements at the PVT1 locus activate additional oncogenic pathways that synergize with MYC to exacerbate the aggressiveness of MYC+ cancers. This newfound understanding explains the poor prognosis associated with MYC+ cancers and offers potential therapeutic targets that could be leveraged in treatment strategies for these cancers.
    DOI:  https://doi.org/10.1101/2025.02.17.638454
  8. Nat Commun. 2025 Mar 04. 16(1): 2191
    A metabolic synthetic lethality of phosphoinositide 3-kinase-driven cancer.
    Guillaume P Andrieu, Mathieu Simonin, Aurélie Cabannes-Hamy, Etienne Lengliné, Ambroise Marçais, Alexandre Théron, Grégoire Huré, Jérome Doss, Ivan Nemazanyy, Marie Émilie Dourthe, Nicolas Boissel, Hervé Dombret, Philippe Rousselot, Olivier Hermine, Vahid Asnafi.
      The deregulated activation of the phosphoinositide 3-kinase (PI3K) pathway is a hallmark of aggressive tumors with metabolic plasticity, eliciting their adaptation to the microenvironment and resistance to chemotherapy. A significant gap lies between the biological features of PI3K-driven tumors and the specific targeting of their vulnerabilities. Here, we explore the metabolic liabilities of PI3K-altered T-cell acute lymphoblastic leukemia (T-ALL), an aggressive hematological cancer with dismal outcomes. We report a metabolic crosstalk linking glutaminolysis and glycolysis driven by PI3K signaling alterations. Pharmaceutical inhibition of mTOR reveals the singular plasticity of PI3K-altered cells toward the mobilization of glutamine as a salvage pathway to ensure their survival. Subsequently, the combination of glutamine degradation and mTOR inhibition demonstrates robust cytotoxicity in PI3K-driven solid and hematological tumors in pre-clinical and clinical settings. We propose a novel therapeutic strategy to circumvent metabolic adaptation and efficiently target PI3K-driven cancer.
    DOI:  https://doi.org/10.1038/s41467-025-57225-7
  9. Bio Protoc. 2025 Feb 20. 15(4): e5191
    Precise Generation of Human Induced Pluripotent Stem Cell-derived Cell Lines Harboring Disease-relevant Single Nucleotide Variants Using a Prime Editing System.
    Seiya Kanno, Kota Sato, Toru Nakazawa.
      Human induced pluripotent stem (iPS) cell lines harboring mutations in disease-related genes serve as invaluable in vitro models for unraveling disease mechanisms and accelerating drug discovery efforts. Introducing mutations into iPS cells using traditional gene editing approaches based on the CRISPR-Cas9 endonuclease often encounters challenges such as unintended insertions/deletions (indels) and off-target effects. To address these limitations, we present a streamlined protocol for introducing highly accurate gene mutations into human iPS cells using prime editing, a "search-and-replace" genome-editing technology that combines unwanted indel-minimized CRISPR-Cas9 nickase with reverse transcriptase. This protocol encompasses the design of prime editing guide RNAs (pegRNAs) required for binding and replacement at target loci, construction of prime editor and pegRNA expression vectors, gene transfer into iPS cells, and cell line selection. This protocol allows for the efficient establishment of disease-associated gene variants within 6-8 weeks while preserving critical genomic context. Key features • Dramatic improvement in efficiency of In-Fusion cloning using inserts assembled from the three pegRNA components (spacer, spCas9 scaffold, and 3' extension) via overlap extension PCR. • Cost-effective and time-saving selection of pegRNAs for prime editing via bulk Sanger sequencing. • Straightforward gene transfection using polymer-based reagents, which requires no specialized equipment or techniques and offers high reproducibility and broad applicability across different cell lines. • Precise genome editing based on pegRNA/prime editing minimizes off-target effects, enabling a wide range of applications in the study of disease-associated genetic variants. Graphical overview Key steps of generation of human induced pluripotent stem (iPS) cell lines harboring disease-relevant single nucleotide variants (SNVs) using a prime editing system.
    Keywords:  CRISPR/Cas9; Disease modeling; Disease-associated single nucleotide variant (SNV); Human induced pluripotent stem (iPS) cell; Isogenic cell line; Precision genome editing; Prime editing; pegRNA design
    DOI:  https://doi.org/10.21769/BioProtoc.5191
  10. Nat Rev Immunol. 2025 Mar 05.
    PerturbView: scalable image-based perturbation screens in cells and tissues.
    Takamasa Kudo.
      
    DOI:  https://doi.org/10.1038/s41577-025-01153-6
  11. bioRxiv. 2025 Feb 17. pii: 2025.02.14.638177. [Epub ahead of print]
    Sex Differences in VEGF and PDGF Ligand and Receptor Protein Expression during Adipose Tissue Expansion and Regression.
    Yingye Fang, Silvia Gonzales-Nieves, Vincenza Cifarelli, P I Imoukhuede.
      Inadequate angiogenesis in obesogenic adipose tissue (AT) has been implicated in disrupted adipogenesis and metabolic disorders. Yet, key cellular and molecular regulators of AT angiogenesis remain largely unidentified. This study sought to identify the dysregulated elements within the Vascular Endothelial Growth Factor (VEGF) and Platelet-Derived Growth Factor (PDGF) systems during obesity progression. We employ a mouse model, comprising both male and female mice, to investigate the changes in the VEGF/PDGF concentration and their receptor distribution in AT during short- and long-term weight gain and weight loss. Our results reveal pronounced sex-specific differences in obesity progression, with male and female mice exhibiting distinct angiogenic ligand and receptor profiles under identical dietary interventions. This data also lays the groundwork for developing computational models of VEGF/PDGF signaling networks in AT, allowing for the simulation of complex biological interactions and the prediction of therapeutic outcomes.
    DOI:  https://doi.org/10.1101/2025.02.14.638177
  12. Sci Adv. 2025 Mar 07. 11(10): eads2998
    Genetically encoded tension heterogeneity sculpts cardiac trabeculation.
    Jinxiu Liang, Peijun Jiang, Shuaifang Yan, Tao Cheng, Shuo Chen, Kexin Xian, Pengfei Xu, Jing-Wei Xiong, Aibin He, Jia Li, Peidong Han.
      The myocardial wall arises from a single layer of cardiomyocytes, some delaminate to create trabeculae while others remain in the compact layer. However, the mechanisms governing cardiomyocyte fate decisions remain unclear. Using single-cell RNA sequencing, genetically encoded biosensors, and in toto live imaging, we observe intrinsic variations in erbb2 expression and its association with trabecular fate. Specifically, erbb2 promotes PI3K activity and recruits the Arp2/3 complex, inducing a polarized accumulation of the actomyosin network to drive cell delamination. Subsequently, the lineage-committed nascent trabeculae trigger Notch activity in neighboring cardiomyocytes to suppress erbb2 expression and reduce cell tension, thereby confining them to the compact layer. Overall, this genetic and cellular interplay governs compact and trabecular cell fate determination to orchestrate myocardial pattern formation.
    DOI:  https://doi.org/10.1126/sciadv.ads2998
  13. Nat Protoc. 2025 Mar 04.
    Unraveling cell-cell communication with NicheNet by inferring active ligands from transcriptomics data.
    Chananchida Sang-Aram, Robin Browaeys, Ruth Seurinck, Yvan Saeys.
      Ligand-receptor interactions constitute a fundamental mechanism of cell-cell communication and signaling. NicheNet is a well-established computational tool that infers ligand-receptor interactions that potentially regulate gene expression changes in receiver cell populations. Whereas the original publication delves into the algorithm and validation, this paper describes a best practices workflow cultivated over four years of experience and user feedback. Starting from the input single-cell expression matrix, we describe a 'sender-agnostic' approach that considers ligands from the entire microenvironment and a 'sender-focused' approach that considers ligands only from cell populations of interest. As output, users will obtain a list of prioritized ligands and their potential target genes, along with multiple visualizations. We include further developments made in NicheNet v2, in which we have updated the data sources and implemented a downstream procedure for prioritizing cell type-specific ligand-receptor pairs. Although a standard NicheNet analysis takes <10 min to run, users often invest additional time in making decisions about the approach and parameters that best suit their biological question. This paper serves to aid in this decision-making process by describing the most appropriate workflow for common experimental designs like case-control and cell-differentiation studies. Finally, in addition to the step-by-step description of the code, we also provide wrapper functions that enable the analysis to be run in one line of code, thus tailoring the workflow to users at all levels of computational proficiency.
    DOI:  https://doi.org/10.1038/s41596-024-01121-9
  14. Cancer Cell. 2025 Feb 20. pii: S1535-6108(25)00053-4. [Epub ahead of print]
    FAK inhibition combined with the RAF-MEK clamp avutometinib overcomes resistance to targeted and immune therapies in BRAF V600E melanoma.
    Simone Lubrano, Rodolfo Daniel Cervantes-Villagrana, Farhoud Faraji, Sydney Ramirez, Kuniaki Sato, Sendi R Adame-Garcia, Adam Officer, Nadia Arang, Damiano C Rigiracciolo, Paola Y Anguiano Quiroz, Claudia Martini, YiYu Wang, Fleur M Ferguson, Antonietta Bacchiocchi, Ruth Halaban, Silvia Coma, Sheri L Holmen, Jonathan A Pachter, Andrew E Aplin, J Silvio Gutkind.
      Widespread BRAF mutations result in persistent RAS-RAF-MEK-ERK (MAPK) signaling in melanoma. BRAF (BRAFi) and MEK (MEKi) inhibitors are approved for BRAF V600E melanomas, including those progressing on immunotherapy; however, rapid resistance to these agents highlights the need for novel strategies. Here, transcriptome analysis of BRAF V600E melanomas from patients resistant to BRAFi and MEKi shows activation of focal adhesion signaling. Consistently, BRAFi, MEKi, and the RAF-MEK clamp avutometinib activate focal adhesion kinase (FAK) in melanoma cells. Mechanistically, inhibition of an MAPK-RhoE (RND3) feedback loop results in the adaptive activation of RhoA-FAK-AKT. In turn, FAK inhibitors (FAKi) exert potent pro-apoptotic activity when combined with MAPK pathway inhibition. FAKi plus avutometinib overcomes resistance in multiple models derived from BRAFi plus MEKi-resistant melanoma patients and immunotherapy-resistant syngeneic mouse models. These findings provide a rationale for the development of avutometinib in combination with FAKi for patients with BRAF V600E melanoma progressing on BRAFi plus MEKi or immunotherapy.
    Keywords:  BRAF; FAK; MAPK signaling; MEK; avutometinib; immunotherapy; melanoma; resistance; signal transduction; targeted therapies
    DOI:  https://doi.org/10.1016/j.ccell.2025.02.001
  15. Nat Struct Mol Biol. 2025 Mar 03.
    Guidelines for minimal reporting requirements, design and interpretation of experiments involving the use of eukaryotic dual gene expression reporters (MINDR).
    Gary Loughran, Dmitry E Andreev, Ilya M Terenin, Olivier Namy, Martin Mikl, Martina M Yordanova, C Joel McManus, Andrew E Firth, John F Atkins, Christopher S Fraser, Zoya Ignatova, Shintaro Iwasaki, Joanna Kufel, Ola Larsson, Sebastian A Leidel, Alexander S Mankin, Marco Mariotti, Marvin E Tanenbaum, Ivan Topisirovic, Nora Vázquez-Laslop, Gabriela Viero, Neva Caliskan, Yiwen Chen, Patricia L Clark, Jonathan D Dinman, Philip J Farabaugh, Wendy V Gilbert, Pavel Ivanov, Jeffrey S Kieft, Oliver Mühlemann, Matthew S Sachs, Ivan N Shatsky, Nahum Sonenberg, Anna-Lena Steckelberg, Anne E Willis, Michael T Woodside, Leos Shivaya Valasek, Sergey E Dmitriev, Pavel V Baranov.
      Dual reporters encoding two distinct proteins within the same mRNA have had a crucial role in identifying and characterizing unconventional mechanisms of eukaryotic translation. These mechanisms include initiation via internal ribosomal entry sites (IRESs), ribosomal frameshifting, stop codon readthrough and reinitiation. This design enables the expression of one reporter to be influenced by the specific mechanism under investigation, while the other reporter serves as an internal control. However, challenges arise when intervening test sequences are placed between these two reporters. Such sequences can inadvertently impact the expression or function of either reporter, independent of translation-related changes, potentially biasing the results. These effects may occur due to cryptic regulatory elements inducing or affecting transcription initiation, splicing, polyadenylation and antisense transcription as well as unpredictable effects of the translated test sequences on the stability and activity of the reporters. Unfortunately, these unintended effects may lead to misinterpretation of data and the publication of incorrect conclusions in the scientific literature. To address this issue and to assist the scientific community in accurately interpreting dual-reporter experiments, we have developed comprehensive guidelines. These guidelines cover experimental design, interpretation and the minimal requirements for reporting results. They are designed to aid researchers conducting these experiments as well as reviewers, editors and other investigators who seek to evaluate published data.
    DOI:  https://doi.org/10.1038/s41594-025-01492-x
  16. Cell Metab. 2025 Mar 04. pii: S1550-4131(25)00001-4. [Epub ahead of print]37(3): 723-741.e6
    A cellular and molecular basis of leptin resistance.
    Bowen Tan, Kristina Hedbacker, Leah Kelly, Zhaoyue Zhang, Alexandre Moura-Assis, Ji-Dung Luo, Joshua D Rabinowitz, Jeffrey M Friedman.
      Similar to most humans with obesity, diet-induced obese (DIO) mice have high leptin levels and fail to respond to the exogenous hormone, suggesting that their obesity is caused by leptin resistance, the pathogenesis of which is unknown. We found that leptin treatment reduced plasma levels of leucine and methionine, mTOR-activating ligands, leading us to hypothesize that chronic mTOR activation might reduce leptin signaling. Rapamycin, an mTOR inhibitor, reduced fat mass and increased leptin sensitivity in DIO mice but not in mice with defects in leptin signaling. Rapamycin restored leptin's actions on POMC neurons and failed to reduce the weight of mice with defects in melanocortin signaling. mTOR activation in POMC neurons caused leptin resistance, whereas POMC-specific mutations in mTOR activators decreased weight gain of DIO mice. Thus, increased mTOR activity in POMC neurons is necessary and sufficient for the development of leptin resistance in DIO mice, establishing a key pathogenic mechanism leading to obesity.
    Keywords:  POMC; diet-induced obesity; leptin; leptin resistance; mTOR; rapamycin
    DOI:  https://doi.org/10.1016/j.cmet.2025.01.001
  17. Brain. 2025 Mar 03. pii: awaf081. [Epub ahead of print]
    TSC2 loss in neural progenitor cells suppresses mRNA translation of neurodevelopmental genes.
    Pauline Martin, Krzysztof J Szkop, Francis Robert, Srirupa Bhattacharyya, Roberta L Beauchamp, Jacob Brenner, Nicholas E Redmond, Sidong Huang, Serkan Erdin, Ola Larsson, Vijaya Ramesh.
      Tuberous sclerosis complex (TSC) is an inherited multi-system neurocutaneous disorder where patients often present with neurodevelopmental manifestations such as epilepsy and TSC-associated neuropsychiatric disorder (TAND) that includes autism spectrum disorder (ASD). TSC is caused by inactivating mutations in TSC1 or TSC2 tumor suppressor genes, with encoded proteins hamartin (TSC1) and tuberin (TSC2) forming a functional complex inhibiting mechanistic target of rapamycin complex 1 (mTORC1) signaling. This has led to treatment with allosteric mTORC1 inhibitor rapamycin analogs ("rapalogs") for TSC tumors; however, rapalogs are ineffective for treating neurodevelopmental manifestations. mTORC1 signaling controls protein synthesis by regulating formation of the eIF4F complex, with further modulation by MNK1/2 kinases via phosphorylation of the eIF4F subunit eIF4E. While both these pathways modulate translation, comparing their impact on transcriptome-wide mRNA translation, as well as effects of inhibiting these pathways in TSC has not been explored. Employing CRISPR-modified, isogenic neural progenitor cells (NPCs) derived from a female TSC2 patient, we have examined alterations in early neurodevelopmental phenotypes including proliferation and neurite outgrowth, as well as ability of bi-steric mTORC1-specific inhibitor RMC-6272 to rescue these phenotypes. Further, we utilized polysome-profiling to examine transcriptome-wide changes in mRNA translation upon TSC2 loss and tested effects of treatment with RMC-6272 or MNK1/2-specific inhibitor eFT-508. Our results reveal that altered early neurodevelopmental phenotypes can be rescued upon treatment with RMC-6272, but not rapamycin. We also discovered dysregulated mRNA translation in TSC2-Null NPCs, which significantly overlaps with the translatome from TSC1-Null NPCs. Interestingly, numerous non-monogenic ASD-, NDD- and epilepsy-associated genes identified in patients harboring putative loss-of-function mutations, were translationally suppressed in TSC2-Null NPCs. Importantly, translation of these ASD- and NDD-associated genes was reversed upon inhibition of either mTORC1 or MNK1/2 signaling using RMC-6272 or eFT-508, respectively. This study establishes the importance of mTORC1-eIF4F- and MNK-eIF4E-sensitive mRNA translation in TAND, ASD and other neurodevelopmental disorders laying the groundwork for evaluating drugs in clinical development that target these pathways as a treatment strategy for these disorders.
    Keywords:  4E-BP1; 5’UTR; CNS; S6K1; protein synthesis; rapalogs
    DOI:  https://doi.org/10.1093/brain/awaf081
  18. bioRxiv. 2025 Feb 17. pii: 2025.02.13.638099. [Epub ahead of print]
    Clonal memory of colitis accumulates and promotes tumor growth.
    Surya Nagaraja, Lety Ojeda-Miron, Ruochi Zhang, Ena Oreskovic, Yan Hu, Daniel Zeve, Karina Sharma, Roni R Hyman, Qiming Zhang, Andrew Castillo, David T Breault, Ömer H Yilmaz, Jason D Buenrostro.
      Chronic inflammation is a well-established risk factor for cancer, but the underlying molecular mechanisms remain unclear. Using a mouse model of colitis, we demonstrate that colonic stem cells retain an epigenetic memory of inflammation following disease resolution, characterized by a cumulative gain of activator protein 1 (AP-1) transcription factor activity. Further, we develop SHARE-TRACE, a method that enables simultaneous profiling of gene expression, chromatin accessibility and clonal history in single cells, enabling high resolution tracking of epigenomic memory. This reveals that inflammatory memory is propagated cell-intrinsically and inherited through stem cell lineages, with certain clones demonstrating dramatically stronger memory than others. Finally, we show that colitis primes stem cells for amplified expression of regenerative gene programs following oncogenic mutation that accelerate tumor growth. This includes a subpopulation of tumors that have exceptionally high AP-1 activity and the additional upregulation of pro-oncogenic programs. Together, our findings provide a mechanistic link between chronic inflammation and malignancy, revealing how long-lived epigenetic alterations in regenerative tissues may contribute to disease susceptibility and suggesting potential therapeutic strategies to mitigate cancer risk in patients with chronic inflammatory conditions.
    DOI:  https://doi.org/10.1101/2025.02.13.638099
  19. J Proteome Res. 2025 Mar 02.
    Affinity Purification Mass Spectrometry on the Orbitrap-Astral Mass Spectrometer Enables High-Throughput Protein-Protein Interaction Mapping.
    Lia R Serrano, Adrian Pelin, Tabiwang N Arrey, Nicolaie E Damoc, Alicia L Richards, Yuan Zhou, Noah M Lancaster, Trenton M Peters-Clarke, Anna Pashkova, Gwendolyn M Jang, Manon Eckhardt, Scott T Quarmby, Martin Zeller, Daniel Hermanson, Hamish Stewart, Christian Hock, Alexander Makarov, Vlad Zabrouskov, Nevan J Krogan, Joshua J Coon, Danielle L Swaney.
      Classical proteomics experiments offer high-throughput protein quantification but lack direct evidence of the spatial organization of the proteome, including protein-protein interaction (PPIs) networks. While affinity purification mass spectrometry (AP-MS) is the method of choice for generating these networks, technological impediments have stymied the throughput of AP-MS sample collection and therefore constrained the rate and scale of experiments that can be performed. Here, we build on advances in mass spectrometry hardware that have rendered high-flow liquid chromatography separations a viable solution for faster throughput quantitative proteomics. We describe our methodology using the Orbitrap-Astral mass spectrometer with 7 min, high-flow separations to analyze 216 AP-MS samples in ∼29 h. We show that the ion-focusing advancements, rapid mass analysis, and sensitive ion detection facilitate narrow-bin data-independent acquisition on a chromatographically practical timescale. Further, we highlight several aspects of state-of-the-art confidence-scoring software that warrant reinvestigation given the analytical characteristics of the Orbitrap-Astral mass spectrometer through comparisons with an enrichment-based thresholding technique. With our data, we generated an interaction map between 998 human proteins and 59 viral proteins. These results hold promise in expediting the throughput of AP-MS experiments, enabling more high-powered PPI studies.
    Keywords:  AP-MS; Astral; PPIs; host–pathogen
    DOI:  https://doi.org/10.1021/acs.jproteome.4c01040
  20. Redox Biol. 2025 Feb 12. pii: S2213-2317(25)00062-X. [Epub ahead of print]81 103549
    Considerations for antibody-based detection of NRF2 in human cells.
    Alicja Dziadosz-Brzezińska, Sara Kusiński, Artur Piróg, Zuzanna Urban-Wójciuk, Monikaben Padariya, Umesh Kalathiya, Sachin Kote, Alicja Sznarkowska.
      Based on the knockdown and overexpression experiments, it is accepted that in Tris-glycine SDS-PAGE human NRF2 migrates above 100 kDa, depending on the percentage of the gel. In 8 % Tris-glycine gel, monoclonal anti-NRF2 antibodies detect NRF2 signal as three bands migrating between 100 and 130 kDa. Here we used mass spectrometry to identify proteins immunoprecipitated by anti-NRF2 antibodies migrating in this range under steady state, upon NRF2 activator tert-BHQ and after translation inhibition with emetine. Our results show that three commercial monoclonal antibodies with epitopes in the center and in the C-terminus of NRF2 also bind calmegin, an ER-residing chaperone, that co-migrates with NRF2 in SDS-PAGE and gives stronger signal in western blot than NRF2. Calmegin has a much longer half life than NRF2 and resides in the cytoplasm, which differentiates it from NRF2. The most specific anti-NRF2 antibody in western blot, Cell Signaling Technology clone E5F1 is also specific in staining nuclear NRF2 in immunofluorescence. Other antibodies, that recognize calmegin in western blot, still can be specific for nuclear NRF2 in immunofluorescence, but require prior validation with NRF2 knockdown or knockout. These results appeal for caution and consideration when analyzing and interpreting results from antibody-based NRF2 detection.
    Keywords:  Calmegin; Immunofluorescence; Mass spectrometry; Monoclonal antibodies; NRF2
    DOI:  https://doi.org/10.1016/j.redox.2025.103549
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