bims-pideca 2025-02-16 papers

bims-pideca

Biomed News

on Class IA PI3K signalling in development and cancer

Issue of 2025–02–16
33 papers selected by
Ralitsa Radostinova Madsen, MRC-PPU

Single-molecule lipid biosensors mitigate inhibition of endogenous effector proteins.
Ezrin defines TSC complex activation at endosomal compartments through EGFR-AKT signaling.
Dysfunctional mechanotransduction regulates the progression of PIK3CA-driven vascular malformations.
A phase 2 randomized, double-blind trial of ART-001, a selective PI3Kα inhibitor, for the treatment of slow-flow vascular malformations.
Regulation and function of insulin and insulin-like growth factor receptor signalling.
Lack of PTEN in osteocytes increases Lipocalin-2 level and confers resistance to high-fat diet-induced obesity in mice.
Polarity-JaM: an image analysis toolbox for cell polarity, junction and morphology quantification.
CRISPuRe-seq: pooled screening of barcoded ribonucleoprotein reporters reveals regulation of RNA polymerase III transcription by the integrated stress response via mTOR.
Lactate controls cancer stemness and plasticity through epigenetic regulation.
Cancer and Overgrowth Manifestations of PTEN Hamartoma Tumour Syndrome: Management Recommendations from the International PHTS Consensus Guidelines Working Group.
Clear cell renal carcinoma essentially requires CDKL3 for oncogenesis.
A guide to selecting high-performing antibodies for Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit delta isoform (PPP2R5D) for use in Western Blot, immunoprecipitation and immunofluorescence.
GitHub enables collaborative and reproducible laboratory research.
Interpretable AI for inference of causal molecular relationships from omics data.
Intercellular adhesion boots collective cell migration through elevated membrane tension.
Targeting PI3Kγ in cancer.
MorPhiC Consortium: towards functional characterization of all human genes.
Receptor clustering tunes and sharpens the selectivity of multivalent binding.
Imaging of cellular dynamics from a whole organism to subcellular scale with self-driving, multiscale microscopy.
Macrophage memory emerges from coordinated transcription factor and chromatin dynamics.
EXPERT expands prime editing efficiency and range of large fragment edits.
Isobaric Tagging and Data Independent Acquisition as Complementary Strategies for Proteome Profiling on an Orbitrap Astral Mass Spectrometer.
Pharmacologic activation of integrated stress response kinases inhibits pathologic mitochondrial fragmentation.
Reporting methods for reusability.
Cellpose3: one-click image restoration for improved cellular segmentation.
Towards an interpretable deep learning model of cancer.
Leveraging prior knowledge to infer gene regulatory networks from single-cell RNA-sequencing data.
Mitofusin 2 displays fusion-independent roles in proteostasis surveillance.
Temporal phosphoproteomics reveals circuitry of phased propagation in insulin signaling.
ProteoArk: A One-Pot Proteomics Data Analysis and Visualization Tool for Biologists.
A molecular proximity sensor based on an engineered, dual-component guide RNA.
Workflow for E3 Ligase Ligand Validation for PROTAC Development.
Inositol polyphosphate multikinase regulates Th1 and Th17 cell differentiation by controlling Akt-mTOR signaling.

J Cell Biol. 2025 Mar 03. pii: e202412026. [Epub ahead of print]224(3):

Single-molecule lipid biosensors mitigate inhibition of endogenous effector proteins.

Victoria L Holmes, Morgan M C Ricci, Claire C Weckerly, Michael Worcester, Gerald R V Hammond.

Genetically encoded lipid biosensors uniquely provide real time, spatially resolved kinetic data for lipid dynamics in living cells. Despite clear strengths, these tools have significant drawbacks; most notably, lipid molecules bound to biosensors cannot engage with effectors, potentially inhibiting signaling. Here, we show that although PI 3-kinase (PI3K)-mediated activation of AKT is not significantly reduced in a cell population transfected with a PH-AKT1 PIP3/PI(3,4)P2 biosensor, single cells expressing PH-AKT at visible levels have reduced activation. Tagging endogenous AKT1 with neonGreen reveals its EGF-mediated translocation to the plasma membrane. Co-transfection with the PH-AKT1 or other PIP3 biosensors eliminates this translocation, despite robust recruitment of the biosensors. Inhibition is even observed with PI(3,4)P2-selective biosensor. However, expressing lipid biosensors at low levels, comparable with those of endogenous AKT, produced no such inhibition. Helpfully, these single-molecule biosensors revealed improved dynamic range and kinetic fidelity compared with overexpressed biosensor. This approach represents a noninvasive way to probe spatiotemporal dynamics of PI3K signaling in living cells.

DOI: https://doi.org/10.1083/jcb.202412026
Elife. 2025 Feb 12. pii: RP98523. [Epub ahead of print]13

Ezrin defines TSC complex activation at endosomal compartments through EGFR-AKT signaling.

Giuliana Giamundo, Daniela Intartaglia, Eugenio Del Prete, Elena Polishchuk, Fabrizio Andreone, Marzia Ognibene, Sara Buonocore, Bruno Hay Mele, Francesco Giuseppe Salierno, Jlenia Monfregola, Dario Antonini, Paolo Grumati, Alessandra Eva, Rossella De Cegli, Ivan Conte.

  Endosomes have emerged as major signaling hubs where different internalized ligand-receptor complexes are integrated and the outcome of signaling pathways are organized to regulate the strength and specificity of signal transduction events. Ezrin, a major membrane-actin linker that assembles and coordinates macromolecular signaling complexes at membranes, has emerged recently as an important regulator of lysosomal function. Here, we report that endosomal-localized EGFR/Ezrin complex interacts with and triggers the inhibition of the Tuberous Sclerosis Complex (TSC complex) in response to EGF stimuli. This is regulated through activation of the AKT signaling pathway. Loss of Ezrin was not sufficient to repress TSC complex by EGF and culminated in translocation of TSC complex to lysosomes triggering suppression of mTORC1 signaling. Overexpression of constitutively active EZRINT567D is sufficient to relocalize TSC complex to the endosomes and reactivate mTORC1. Our findings identify EZRIN as a critical regulator of autophagy via TSC complex in response to EGF stimuli and establish the central role of early endosomal signaling in the regulation of mTORC1. Consistently, Medaka fish deficient for Ezrin exhibit defective endo-lysosomal pathway, attributable to the compromised EGFR/AKT signaling, ultimately leading to retinal degeneration. Our data identify a pivotal mechanism of endo-lysosomal signaling involving Ezrin and its associated EGFR/TSC complex, which are essential for retinal function.

Keywords:  EGFR; EZRIN; TSC complex; cell biology; endosome; lysosome; mTORC1

DOI:  https://doi.org/10.7554/eLife.98523
APL Bioeng. 2025 Mar;9(1): 016106

Dysfunctional mechanotransduction regulates the progression of PIK3CA-driven vascular malformations.

Wen Yih Aw, Aanya Sawhney, Mitesh Rathod, Chloe P Whitworth, Elizabeth L Doherty, Ethan Madden, Jingming Lu, Kaden Westphal, Ryan Stack, William J Polacheck.

Somatic activating mutations in PIK3CA are common drivers of vascular and lymphatic malformations. Despite common biophysical signatures of tissues susceptible to lesion formation, including compliant extracellular matrix and low rates of perfusion, lesions vary in clinical presentation from localized cystic dilatation to diffuse and infiltrative vascular dysplasia. The mechanisms driving the differences in disease severity and variability in clinical presentation and the role of the biophysical microenvironment in potentiating progression are poorly understood. Here, we investigate the role of hemodynamic forces and the biophysical microenvironment in the pathophysiology of vascular malformations (VMs), and we identify hemodynamic shear stress and defective endothelial cell mechanotransduction as key regulators of lesion progression. We found that constitutive PI3K activation impaired flow-mediated endothelial cell alignment and barrier function. We show that defective shear stress sensing in PIK3CAE542K endothelial cells is associated with reduced myosin light chain phosphorylation, junctional instability, and defective recruitment of vinculin to cell-cell junctions. Using three dimensional (3D) microfluidic models of the vasculature, we demonstrate that PIK3CAE542K microvessels apply reduced traction forces and are unaffected by flow interruption. We further found that draining transmural flow resulted in increased sprouting and invasion responses in PIK3CAE542K microvessels. Mechanistically, constitutive PI3K activation decreased cellular and nuclear elasticity resulting in defective cellular tensional homeostasis in endothelial cells which may underlie vascular dilation, tissue hyperplasia, and hypersprouting in PIK3CA-driven venous and lymphatic malformations. Together, these results suggest that defective nuclear mechanics, impaired cellular mechanotransduction, and maladaptive hemodynamic responses contribute to the development and progression of PIK3CA-driven vascular malformations.

DOI: https://doi.org/10.1063/5.0234507
Orphanet J Rare Dis. 2025 Feb 10. 20(1): 64

A phase 2 randomized, double-blind trial of ART-001, a selective PI3Kα inhibitor, for the treatment of slow-flow vascular malformations.

Michio Ozeki, Akira Tanaka, Kanako Kuniyeda, Taiki Nozaki, Akihiro Fujino, Tadashi Nomura, Naoto Uemura, Souichi Suenobu, Noriko Aramaki-Hattori, Ayato Hayashi, Aiko Kato, Hiro Kiyosue, Kotaro Imagawa, Munetomo Nagao, Fumiaki Shimizu, Junko Ochi, Saya Horiuchi, Tetsuji Ohyama, Haruhi Ando, Hiroshi Nagabukuro.

   BACKGROUND: In patients with slow-flow vascular malformations (SFVMs) including venous malformations (VM), lymphatic malformations (LM) or Klippel-Trenaunay Syndrome (KTS), somatic gain-of-function mutations in genes encoding phosphatidyl inositol 3-kinase alpha (PI3Kα, gene name PIK3CA) have been identified. A phase 2 study was conducted with the patients to assess the efficacy and safety of ART-001 (serabelisib), an orally available selective PI3Kα inhibitor.
METHODS: This is a multicenter, randomized, double-blind, proof-of-concept, phase 2 trial. Eligible participants were patients aged 2 years and older, diagnosed either with VM, LM or KTS. Participants were administered either 50 or 100 mg of ART-001 for 24 weeks. The primary endpoint was the response rate defined as the proportion of participants who achieved ≥ 20% reduction in lesion volume at week 24. Secondary endpoints include safety, pharmacokinetics, pain, and quality of life scores.
RESULTS: Thirty-five patients (median age: 14 years old; VM, n = 17, KTS, n = 13 and LM, n = 5) were randomly assigned and received treatment (50 mg, n = 17 and 100 mg, n = 18). ART-001 showed a response rate: 29.4% (95% confidence interval 10.3-56.0%) at 50 mg and 33.3% (13.3-59.0%) at 100 mg. Mean lesion volume reductions at 50 mg and 100 mg were - 2.3% (95% CI - 14.3 to 9.6%) and - 12.6% (- 25.3 to 0.06%), respectively. No drug-related serious adverse events were observed. Treatment-emergent adverse events were generally mild to moderate and transient. Pharmacokinetic profiles were similar between pediatric and adolescent/adult patients except for lower Ctrough levels in pediatric patients.
CONCLUSION: ART-001 was effective and well-tolerated in patients with SFVMs. These results support the further development of ART-001 in SFVMs and other PIK3CA-related overgrowth syndromes to confirm clinical benefits and long-term safety.
TRIAL REGISTRATION: Japan Registry of Clinical Trial, jRCT2071210027. Registered May 25 2021, https://jrct.niph.go.jp/en-latest-detail/jRCT2071210027.

Keywords:  And drug development; PI3Kα; Pharmacotherapy; Phase 2 study; Vascular malformations

DOI:  https://doi.org/10.1186/s13023-025-03564-z
Nat Rev Mol Cell Biol. 2025 Feb 10.

Regulation and function of insulin and insulin-like growth factor receptor signalling.

Eunhee Choi, Cunming Duan, Xiao-Chen Bai.

Receptors of insulin and insulin-like growth factors (IGFs) are receptor tyrosine kinases whose signalling controls multiple aspects of animal physiology throughout life. In addition to regulating metabolism and growth, insulin-IGF receptor signalling has recently been linked to a variety of new, cell type-specific functions. In the last century, key questions have focused on how structural differences of insulin and IGFs affect receptor activation, and how insulin-IGF receptor signalling translates into pleiotropic biological functions. Technological advances such as cryo-electron microscopy have provided a detailed understanding of how native and engineered ligands activate insulin-IGF receptors. In this Review, we highlight recent structural and functional insights into the activation of insulin-IGF receptors, and summarize new agonists and antagonists developed for intervening in the activation of insulin-IGF receptor signalling. Furthermore, we discuss recently identified regulatory mechanisms beyond ligand-receptor interactions and functions of insulin-IGF receptor signalling in diseases.

DOI: https://doi.org/10.1038/s41580-025-00826-3
Endocrinology. 2025 Feb 08. pii: bqaf026. [Epub ahead of print]

Lack of PTEN in osteocytes increases Lipocalin-2 level and confers resistance to high-fat diet-induced obesity in mice.

Saori Kinoshita, Shinsuke Onuma, Natsuko Yamazaki, Yukinao Shibukawa, Keiichi Ozono, Toshimi Michigami, Masanobu Kawai.

  Osteocytes have been shown to play critical roles in the regulation of a wide range of metabolic processes. However, their role in the regulation of glucose metabolism remains to be determined despite accumulating evidence of the integral role of osteoblasts in this regulation, in which osteoblast-derived Lipocalin-2 (LCN2) has been shown to regulate glucose metabolism. Additionally, Lcn2 expression is induced by AKT activation. These results led us to hypothesize that AKT activation in osteocytes regulates glucose metabolism by modulating Lcn2 expression. Therefore, in this study, the Pten gene was deleted in osteocytes to activate AKT signaling by crossing Pten-flox mice with Dmp1-Cre mice (PtenOcy-/- mice). Deleting Pten expression in osteocytes resulted in osteocytic AKT activation, which was associated with decreased adipose tissue mass and enhanced insulin sensitivity. Expression of Pparg2 and lipogenesis-associated genes were decreased in the adipose tissue of PtenOcy-/- mice. Mechanistically, the lack of PTEN in osteocytes increased Lcn2 expression in the femur, which was associated with increased serum and urine LCN2 levels. The urinary LCN2 level was negatively associated with white adipose tissue mass. Additionally, the treatment of primary white adipocytes with recombinant LCN2 reduced the expression of Pparg2 and lipogenesis-related genes. These results suggest that the absence of PTEN in osteocytes increases the expression of Lcn2, which acts in the adipose tissue to suppress lipogenesis, resulting in enhanced insulin sensitivity in these mice. This study provides novel insights into the critical role of AKT activation in osteocytes in regulating glucose metabolism by increasing Lcn2 expression.

Keywords:  Lipocalin-2; Osteocyte; PTEN; glucose metabolism

DOI:  https://doi.org/10.1210/endocr/bqaf026
Nat Commun. 2025 Feb 08. 16(1): 1474

Polarity-JaM: an image analysis toolbox for cell polarity, junction and morphology quantification.

Wolfgang Giese, Jan Philipp Albrecht, Olya Oppenheim, Emir Bora Akmeriç, Julia Kraxner, Deborah Schmidt, Kyle Harrington, Holger Gerhardt.

Cell polarity involves the asymmetric distribution of cellular components such as signalling molecules and organelles within a cell, alterations in cell morphology and cell-cell contacts. Advances in fluorescence microscopy and deep learning algorithms open up a wealth of unprecedented opportunities to characterise various aspects of cell polarity, but also create new challenges for comprehensible and interpretable image data analysis workflows to fully exploit these new opportunities. Here we present Polarity-JaM, an open source package for reproducible exploratory image analysis that provides versatile methods for single cell segmentation, feature extraction and statistical analysis. We demonstrate our analysis using fluorescence image data of endothelial cells and their collective behaviour, which has been shown to be essential for vascular development and disease. The general architecture of the software allows its application to other cell types and imaging modalities, as well as seamless integration into common image analysis workflows, see https://polarityjam.readthedocs.io . We also provide a web application for circular statistics and data visualisation, available at www.polarityjam.com , and a Napari plug-in, each with a graphical user interface to facilitate exploratory analysis. We propose a holistic image analysis workflow that is accessible to the end user in bench science, enabling comprehensive analysis of image data.

DOI: https://doi.org/10.1038/s41467-025-56643-x
Nucleic Acids Res. 2025 Feb 08. pii: gkaf062. [Epub ahead of print]53(4):

CRISPuRe-seq: pooled screening of barcoded ribonucleoprotein reporters reveals regulation of RNA polymerase III transcription by the integrated stress response via mTOR.

David T Harris, Calvin H Jan.

Genetic screens using CRISPR (Clustered Regularly Interspaced Palindromic Repeats) provide valuable information about gene function. Nearly all pooled screening technologies rely on the cell to link genotype to phenotype, making it challenging to assay mechanistically informative, biochemically defined phenotypes. Here, we present CRISPuRe-seq (CRISPR PuRification), a novel pooled screening strategy that expands the universe of accessible phenotypes through the purification of ribonucleoprotein complexes that link genotypes to expressed RNA barcodes. While screening for regulators of the integrated stress response (ISR), we serendipitously discovered that the ISR represses transfer RNA (tRNA) production under conditions of reduced protein synthesis. This regulation is mediated through inhibition of mTORC1 and corresponding activation of the RNA polymerase III inhibitor MAF1. These data demonstrate that coherent downregulation of tRNA expression and protein synthesis is achieved through cross-talk between the ISR and mTOR, two master integrators of cell state.

DOI: https://doi.org/10.1093/nar/gkaf062
Cell Metab. 2025 Feb 04. pii: S1550-4131(25)00002-6. [Epub ahead of print]

Lactate controls cancer stemness and plasticity through epigenetic regulation.

Nguyen T B Nguyen, Sira Gevers, Rutger N U Kok, Lotte M Burgering, Hannah Neikes, Ninouk Akkerman, Max A Betjes, Marlies C Ludikhuize, Can Gulersonmez, Edwin C A Stigter, Yvonne Vercoulen, Jarno Drost, Hans Clevers, Michiel Vermeulen, Jeroen S van Zon, Sander J Tans, Boudewijn M T Burgering, Maria J Rodríguez Colman.

  Tumors arise from uncontrolled cell proliferation driven by mutations in genes that regulate stem cell renewal and differentiation. Intestinal tumors, however, retain some hierarchical organization, maintaining both cancer stem cells (CSCs) and cancer differentiated cells (CDCs). This heterogeneity, coupled with cellular plasticity enabling CDCs to revert to CSCs, contributes to therapy resistance and relapse. Using genetically encoded fluorescent reporters in human tumor organoids, combined with our machine-learning-based cell tracker, CellPhenTracker, we simultaneously traced cell-type specification, metabolic changes, and reconstructed cell lineage trajectories during tumor organoid development. Our findings reveal distinctive metabolic phenotypes in CSCs and CDCs. We find that lactate regulates tumor dynamics, suppressing CSC differentiation and inducing dedifferentiation into a proliferative CSC state. Mechanistically, lactate increases histone acetylation, epigenetically activating MYC. Given that lactate's regulation of MYC depends on the bromodomain-containing protein 4 (BRD4), targeting cancer metabolism and BRD4 inhibitors emerge as a promising strategy to prevent tumor relapse.

Keywords:  cancer metabolism; cell plasticity; cell types; cell-cell interactions; differentiation; heterogeneity; live imaging; organoids; single-cell tracking; stem cells

DOI:  https://doi.org/10.1016/j.cmet.2025.01.002
Clin Cancer Res. 2025 Feb 12.

Cancer and Overgrowth Manifestations of PTEN Hamartoma Tumour Syndrome: Management Recommendations from the International PHTS Consensus Guidelines Working Group.

Andrew Dhawan, Sarah Baitamouni, Darren Liu, Lamis Yehia, Kristin Anthony, Alicia McCarther, Marc Tischkowitz, Suzanne P MacFarland, Joanne Ngeow, Nicoline Hoogerbrugge, Charis Eng.

PURPOSE: PTEN hamartoma tumour syndrome (PHTS) is an autosomal dominant cancer-predisposition and overgrowth syndrome occurring due to pathogenic germline variants in the PTEN gene, with an increased risk of both benign and malignant tumours involving the breast, colon, endometrium, thyroid, skin, and kidney. The objective of these clinical guidelines was to use the latest knowledge to generate an international consensus resource for providers, researchers, and individuals with PHTS on the best practices in the surveillance and management of cancer and overgrowth in PHTS.
EXPERIMENTAL DESIGN: The International PHTS Cancer and Overgrowth Guidelines Working Group was established, comprising a core group of six international experts in the diagnosis and management of PHTS. The Working Group held joint meetings with PHTS individuals and their advocates. Informed by the literature, the Working Group met regularly between 2022 and 2024 to produce guideline statements, refined through iterative feedback. A modified Delphi approach was used with an independent external panel of PHTS, genetics and cancer experts to establish final consensus guidelines.
RESULTS: Clinical consensus recommendations for the surveillance and management of cancer and overgrowth in individuals with PHTS were formed. Guidelines encompass the recommended practices in cases of breast, colon, endometrial, thyroid, and kidney cancers, as well as overgrowths.
CONCLUSIONS: The clinical management of individuals with PHTS is complex and necessitates a multidisciplinary approach. We generated international consensus guidelines for the surveillance and management of cancer and overgrowth in PHTS aiming at improving care for affected individuals and families.

DOI: https://doi.org/10.1158/1078-0432.CCR-24-3819
Proc Natl Acad Sci U S A. 2025 Feb 18. 122(7): e2415244122

Clear cell renal carcinoma essentially requires CDKL3 for oncogenesis.

Lanjing Ma, Zhongqiu Pang, Haijiao Zhang, Xueling Yang, Shaoqin Zheng, Yi Chen, Weijie Ding, Qing Han, Xi Zhang, Liu Cao, Teng Fei, Qiang Wang, Daming Gao, Aina He, Ke-Bang Hu, Xuexin Li, Ren Sheng.

  Clear cell renal cell carcinoma (ccRCC) is the predominant human renal cancer with surging incidence and fatality lately. Hyperactivation of hypoxia-inducible factor (HIF) and mammalian target of rapamycin (mTOR) signaling are the common signatures in ccRCC. Herein, we employed spontaneous ccRCC model to demonstrate the indispensability of an underappreciated Ser/Thr kinase, CDKL3, in the initiation and progression of ccRCC. Ablation of CDKL3 does not affect normal kidney, but abrogates Akt-mTOR hyperactivity and thoroughly prevents the formation and growth of the HIF-agitated ccRCC in vivo. Remarkable clinical correlations also supported the oncogenic role of CDKL3. Mechanism-wise, cytosolic CDKL3 unexpectedly behaves as the adaptor to physically potentiate mTORC2-dependent Akt activation without functioning through kinase activity. And mTORC2 can phosphorylate and stabilize CDKL3 to form a positive feedback loop to sustain the cancer-favored Akt-mTOR overactivation. Together, we revealed the pathological importance and molecular mechanism of CDKL3-mediated Akt-mTOR axis in ccRCC initiation and progression.

Keywords:  Akt; CDKL3; ccRCC; mTOR; oncogene

DOI:  https://doi.org/10.1073/pnas.2415244122
F1000Res. 2024 ;13 1

A guide to selecting high-performing antibodies for Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit delta isoform (PPP2R5D) for use in Western Blot, immunoprecipitation and immunofluorescence.

NeuroSGC/YCharOS/EDDU collaborative group

  Protein phosphatase 2A is a serine/threonine phosphatase with activity dependent on an associated regulatory subunit, serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit delta (δ) isoform (PPP2R5D). PPP2R5D is the δ isoform in the B56 family of regulatory subunits. Abundantly expressed in the brain and involved in a broad range of cellular processes, PPP2R5D plays an essential role in modulating key neuronal pathways and signalling. Pathogenic mutations in the PPP2R5D gene are linked to clinical symptoms characterized by neurodevelopmental delay, intellectual disability, and autism spectrum disorders. The etiology of these genetic disorders remains unknown, which can partly be due to the lack of independently characterized antibodies. Here we have characterized six PPP2R5D commercial antibodies for Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Keywords:  PPP2R5D; Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit delta isoform; Uniprot ID Q14738; Western Blot; antibody characterization; antibody validation; immunofluorescence; immunoprecipitation

DOI:  https://doi.org/10.12688/f1000research.145146.2
PLoS Biol. 2025 Feb;23(2): e3003029

GitHub enables collaborative and reproducible laboratory research.

Katharine Y Chen, Maria Toro-Moreno, Arvind Rasi Subramaniam.

GitHub, a platform widely used in software development, offers a robust framework for documenting all activities of laboratory research projects. This Community Page highlights the benefits of, and provides guidance for, incorporating the GitHub ecosystem into "wet" lab workflows.

DOI: https://doi.org/10.1371/journal.pbio.3003029
Sci Adv. 2025 Feb 14. 11(7): eadk0837

Interpretable AI for inference of causal molecular relationships from omics data.

Payam Dibaeinia, Abhishek Ojha, Saurabh Sinha.

The discovery of molecular relationships from high-dimensional data is a major open problem in bioinformatics. Machine learning and feature attribution models have shown great promise in this context but lack causal interpretation. Here, we show that a popular feature attribution model, under certain assumptions, estimates an average of a causal quantity reflecting the direct influence of one variable on another. We leverage this insight to propose a precise definition of a gene regulatory relationship and implement a new tool, CIMLA (Counterfactual Inference by Machine Learning and Attribution Models), to identify differences in gene regulatory networks between biological conditions, a problem that has received great attention in recent years. Using extensive benchmarking on simulated data, we show that CIMLA is more robust to confounding variables and is more accurate than leading methods. Last, we use CIMLA to analyze a previously published single-cell RNA sequencing dataset from subjects with and without Alzheimer's disease (AD), discovering several potential regulators of AD.

DOI: https://doi.org/10.1126/sciadv.adk0837
Nat Commun. 2025 Feb 12. 16(1): 1588

Intercellular adhesion boots collective cell migration through elevated membrane tension.

Brent M Bijonowski, Jongkwon Park, Martin Bergert, Christina Teubert, Alba Diz-Muñoz, Milos Galic, Seraphine V Wegner.

In multicellular systems, the migration pattern of individual cells critically relies on the interactions with neighboring cells. Depending on the strength of these interactions, cells either move as a collective, as observed during morphogenesis and wound healing, or migrate individually, as it is the case for immune cells and fibroblasts. Mediators of cell-cell adhesions, such as cadherins coordinate collective dynamics by linking the cytoskeleton of neighboring cells. However, whether intercellular binding alone triggers signals that originate from within the plasma membrane itself, remains unclear. Here, we address this question through artificial photoswitchable cell-cell adhesions that selectively connect adjacent plasma membranes without linking directly to cytoskeletal elements. We find that these intercellular adhesions are sufficient to achieve collective cell migration. Linking adjacent cells increases membrane tension, which activates the enzyme phospholipase D2. The resulting increase in phosphatidic acid, in turn, stimulates the mammalian target of rapamycin, a known actuator of collective cell migration. Collectively, these findings introduce a membrane-based signaling axis as promotor of collective cell dynamics, which is independent of the direct coupling of cell-cell adhesions to the cytoskeleton.

DOI: https://doi.org/10.1038/s41467-025-56941-4
Trends Cancer. 2025 Feb 12. pii: S2405-8033(25)00009-3. [Epub ahead of print]

Targeting PI3Kγ in cancer.

Giuliana P Mognol, Anghesom Ghebremedhin, Judith A Varner.

  The phosphoinositide 3-kinases (PI3Ks) have been the focus of a significant body of cancer research since their discovery nearly 40 years ago. These lipid kinases are now known to play central roles in cancer cell proliferation, survival, migration, metabolism, and immunity and serve as the target of numerous investigational and approved therapeutics. One of these kinases, the unique class IB PI3Kγ, which is highly expressed in myeloid lineage cells and myeloid leukemias, plays prominent roles in tumor immune suppression. Inhibition of this kinase has promoted improved antitumor immune responses in recent solid tumor preclinical studies and clinical trials. New studies also identify this kinase as a driver of acute myeloid leukemia self-renewal and as a new target for the treatment of aggressive leukemias.

Keywords:  PI3Kγ; PIK3CG; acute myeloid leukemia tumor stemness; myeloid cell trafficking; phosphatidylinositol-3 kinase; phosphatidylinositol-3 kinase gamma; phosphoinositide-3 kinase inhibitors; solid tumor inflammation

DOI:  https://doi.org/10.1016/j.trecan.2025.01.008
Nature. 2025 Feb;638(8050): 351-359

MorPhiC Consortium: towards functional characterization of all human genes.

MorPhiC Consortium

Recent advances in functional genomics and human cellular models have substantially enhanced our understanding of the structure and regulation of the human genome. However, our grasp of the molecular functions of human genes remains incomplete and biased towards specific gene classes. The Molecular Phenotypes of Null Alleles in Cells (MorPhiC) Consortium aims to address this gap by creating a comprehensive catalogue of the molecular and cellular phenotypes associated with null alleles of all human genes using in vitro multicellular systems. In this Perspective, we present the strategic vision of the MorPhiC Consortium and discuss various strategies for generating null alleles, as well as the challenges involved. We describe the cellular models and scalable phenotypic readouts that will be used in the consortium's initial phase, focusing on 1,000 protein-coding genes. The resulting molecular and cellular data will be compiled into a catalogue of null-allele phenotypes. The methodologies developed in this phase will establish best practices for extending these approaches to all human protein-coding genes. The resources generated-including engineered cell lines, plasmids, phenotypic data, genomic information and computational tools-will be made available to the broader research community to facilitate deeper insights into human gene functions.

DOI: https://doi.org/10.1038/s41586-024-08243-w
Proc Natl Acad Sci U S A. 2025 Feb 18. 122(7): e2417159122

Receptor clustering tunes and sharpens the selectivity of multivalent binding.

Zhaoping Xie, Stefano Angioletti-Uberti, Jure Dobnikar, Daan Frenkel, Tine Curk.

  The immune system exploits a wide range of strategies to combine sensitivity with selectivity for optimal response. We propose a generic physical mechanism that allows tuning the location and steepness of the response threshold of cellular processes activated by multivalent binding. The mechanism is based on the possibility to modulate the attraction between membrane receptors. We use theory and simulations to show how tuning interreceptor attraction can enhance or suppress the binding of multivalent ligand-coated particles to surfaces. The changes in the interreceptor attraction less than the thermal energy kBT can selectively switch the receptor-clustering and activation on or off in an almost step-wise fashion, which we explain by near-critical receptor density fluctuations. We also show that the same mechanism can efficiently regulate the onset of endocytosis for, e.g., drug delivery vehicles.

Keywords:  Monte Carlo; multivalent binding; receptor clustering; superselectivity

DOI:  https://doi.org/10.1073/pnas.2417159122
Nat Methods. 2025 Feb 12.

Imaging of cellular dynamics from a whole organism to subcellular scale with self-driving, multiscale microscopy.

Stephan Daetwyler, Hanieh Mazloom-Farsibaf, Felix Y Zhou, Dagan Segal, Etai Sapoznik, Bingying Chen, Jill M Westcott, Rolf A Brekken, Gaudenz Danuser, Reto Fiolka.

Most biological processes, from development to pathogenesis, span multiple time and length scales. While light-sheet fluorescence microscopy has become a fast and efficient method for imaging organisms, cells and subcellular dynamics, simultaneous observations across all these scales have remained challenging. Moreover, continuous high-resolution imaging inside living organisms has mostly been limited to a few hours, as regions of interest quickly move out of view due to sample movement and growth. Here, we present a self-driving, multiresolution light-sheet microscope platform controlled by custom Python-based software, to simultaneously observe and quantify subcellular dynamics in the context of entire organisms in vitro and in vivo over hours of imaging. We apply the platform to the study of developmental processes, cancer invasion and metastasis, and we provide quantitative multiscale analysis of immune-cancer cell interactions in zebrafish xenografts.

DOI: https://doi.org/10.1038/s41592-025-02598-2
Cell Syst. 2025 Feb 06. pii: S2405-4712(25)00004-3. [Epub ahead of print] 101171

Macrophage memory emerges from coordinated transcription factor and chromatin dynamics.

Andrew G Wang, Minjun Son, Aleksandr Gorin, Emma Kenna, Abinash Padhi, Bijentimala Keisham, Adam Schauer, Alexander Hoffmann, Savaş Tay.

  Cells of the immune system operate in dynamic microenvironments where the timing, concentration, and order of signaling molecules constantly change. Despite this complexity, immune cells manage to communicate accurately and control inflammation and infection. It is unclear how these dynamic signals are encoded and decoded and if individual cells retain the memory of past exposure to inflammatory molecules. Here, we use live-cell analysis, ATAC sequencing, and an in vivo model of sepsis to show that sequential inflammatory signals induce memory in individual macrophages through reprogramming the nuclear factor κB (NF-κB) network and the chromatin accessibility landscape. We use transcriptomic profiling and deep learning to show that transcription factor and chromatin dynamics coordinate fine-tuned macrophage responses to new inflammatory signals. This work demonstrates how macrophages retain the memory of previous signals despite single-cell variability and elucidates the mechanisms of signal-induced memory in dynamic inflammatory conditions like sepsis.

Keywords:  NF-κB; Toll-like receptors; dynamics; inflammatory signaling; innate immune memory; modeling; sepsis; signaling dynamics; systems biology

DOI:  https://doi.org/10.1016/j.cels.2025.101171
Nat Commun. 2025 Feb 13. 16(1): 1592

EXPERT expands prime editing efficiency and range of large fragment edits.

Youcai Xiong, Yinyu Su, Ruigao He, Xiaosong Han, Sheng Li, Minghuan Liu, Xiaoning Xi, Zijia Liu, Heng Wang, Shengsong Xie, Xuewen Xu, Kui Li, Jifeng Zhang, Jie Xu, Xinyun Li, Shuhong Zhao, Jinxue Ruan.

Prime editing systems (PEs) hold great promise in modern biotechnology. However, their editing range is limited as PEs can only modify the downstream sequences of the pegRNA nick. Here, we report the development of the extended prime editor system (EXPERT) to overcome this limitation by using an extended pegRNA (ext-pegRNA) with modified 3' extension, and an additional sgRNA (ups-sgRNA) targeting the upstream region of the ext-pegRNA. We demonstrate that EXPERT can efficiently perform editing on both sides of the ext-pegRNA nick, a task that is unattainable by canonical PEs. EXPERT exhibits prominent capacity in replacing sequences up to 88 base pairs and inserting sequences up to 100 base pairs within the upstream region of the ext-pegRNA nick. Compared to canonical PEs such as PE2, the utilization of the EXPERT strategy significantly enhances the editing efficiency for large fragment edits with an average improvement of 3.12-fold, up to 122.1 times higher. Safety wise, the use of ups-sgRNA does not increase the rates of undesirable insertions and deletions (indels), as the two nicks are on the same strand. Moreover, we do not observe increased off-target editing rates genome-wide. Our work introduces EXPERT as a PE tool with significant potential in life sciences.

DOI: https://doi.org/10.1038/s41467-025-56734-9
J Proteome Res. 2025 Feb 12.

Isobaric Tagging and Data Independent Acquisition as Complementary Strategies for Proteome Profiling on an Orbitrap Astral Mass Spectrometer.

Xinyue Liu, Shane L Dawson, Steven P Gygi, Joao A Paulo.

  Comprehensive global proteome profiling that is amenable to high throughput processing will broaden our understanding of complex biological systems. Here, we evaluate two leading mass spectrometry techniques, Data Independent Acquisition (DIA) and Tandem Mass Tagging (TMT), for extensive protein abundance profiling. DIA provides label-free quantification with a broad dynamic range, while TMT enables multiplexed analysis using isobaric tags for efficient cross-sample comparisons. We analyzed 18 samples, including four cell lines (IHCF, HCT116, HeLa, MCF7) under standard growth conditions, in addition to IHCF treated with two H2O2 concentrations, all in triplicate. Experiments were conducted on an Orbitrap Astral mass spectrometer, employing Field Asymmetric Ion Mobility Spectrometry (FAIMS). Despite utilizing different acquisition strategies, both the DIA and TMT approaches achieved comparable proteome depth and quantitative consistency, with each method quantifying over 10,000 proteins across all samples, with marginally higher protein-level precision for the TMT strategy. Relative abundance correlation analysis showed strong agreement at both peptide and protein levels. Our findings highlight the complementary strengths of DIA and TMT for high-coverage proteomic studies, providing flexibility in method selection based on specific experimental needs.

Keywords:  Astral; DIA; FAIMS; IHCF; TMTpro

DOI:  https://doi.org/10.1021/acs.jproteome.4c01107
Elife. 2025 Feb 12. pii: RP100541. [Epub ahead of print]13

Pharmacologic activation of integrated stress response kinases inhibits pathologic mitochondrial fragmentation.

Kelsey R Baron, Samantha Oviedo, Sophia Krasny, Mashiat Zaman, Rama Aldakhlallah, Prerona Bora, Prakhyat Mathur, Gerald Pfeffer, Michael J Bollong, Timothy E Shutt, Danielle A Grotjahn, R Luke Wiseman.

  Excessive mitochondrial fragmentation is associated with the pathologic mitochondrial dysfunction implicated in the pathogenesis of etiologically diverse diseases, including many neurodegenerative disorders. The integrated stress response (ISR) - comprising the four eIF2α kinases PERK, GCN2, PKR, and HRI - is a prominent stress-responsive signaling pathway that regulates mitochondrial morphology and function in response to diverse types of pathologic insult. This suggests that pharmacologic activation of the ISR represents a potential strategy to mitigate pathologic mitochondrial fragmentation associated with human disease. Here, we show that pharmacologic activation of the ISR kinases HRI or GCN2 promotes adaptive mitochondrial elongation and prevents mitochondrial fragmentation induced by the calcium ionophore ionomycin. Further, we show that pharmacologic activation of the ISR reduces mitochondrial fragmentation and restores basal mitochondrial morphology in patient fibroblasts expressing the pathogenic D414V variant of the pro-fusion mitochondrial GTPase MFN2 associated with neurological dysfunctions, including ataxia, optic atrophy, and sensorineural hearing loss. These results identify pharmacologic activation of ISR kinases as a potential strategy to prevent pathologic mitochondrial fragmentation induced by disease-relevant chemical and genetic insults, further motivating the pursuit of highly selective ISR kinase-activating compounds as a therapeutic strategy to mitigate mitochondrial dysfunction implicated in diverse human diseases.

Keywords:  cell biology; human; integrated stress response; mitochondrial fragmentation; mitochondrial morphology; mouse; stress signaling

DOI:  https://doi.org/10.7554/eLife.100541
Nat Methods. 2025 Feb;22(2): 217

Reporting methods for reusability.

DOI: https://doi.org/10.1038/s41592-025-02615-4
Nat Methods. 2025 Feb 12.

Cellpose3: one-click image restoration for improved cellular segmentation.

Carsen Stringer, Marius Pachitariu.

Generalist methods for cellular segmentation have good out-of-the-box performance on a variety of image types; however, existing methods struggle for images that are degraded by noise, blurring or undersampling, all of which are common in microscopy. We focused the development of Cellpose3 on addressing these cases and here we demonstrate substantial out-of-the-box gains in segmentation and image quality for noisy, blurry and undersampled images. Unlike previous approaches that train models to restore pixel values, we trained Cellpose3 to output images that are well segmented by a generalist segmentation model, while maintaining perceptual similarity to the target images. Furthermore, we trained the restoration models on a large, varied collection of datasets, thus ensuring good generalization to user images. We provide these tools as 'one-click' buttons inside the graphical interface of Cellpose as well as in the Cellpose API.

DOI: https://doi.org/10.1038/s41592-025-02595-5
NPJ Precis Oncol. 2025 Feb 14. 9(1): 46

Towards an interpretable deep learning model of cancer.

Avlant Nilsson, Nikolaos Meimetis, Douglas A Lauffenburger.

Cancer is a manifestation of dysfunctional cell states. It emerges from an interplay of intrinsic and extrinsic factors that disrupt cellular dynamics, including genetic and epigenetic alterations, as well as the tumor microenvironment. This complexity can make it challenging to infer molecular causes for treating the disease. This may be addressed by system-wide computer models of cells, as they allow rapid generation and testing of hypotheses that would be too slow or impossible to perform in the laboratory and clinic. However, so far, such models have been impeded by both experimental and computational limitations. In this perspective, we argue that they can now be achieved using deep learning algorithms to integrate omics data and prior knowledge of molecular networks. Such models would have many applications in precision oncology, e.g., for identifying drug targets and biomarkers, predicting resistance mechanisms and toxicity effects of drugs, or simulating cell-cell interactions in the microenvironment.

DOI: https://doi.org/10.1038/s41698-025-00822-y
Mol Syst Biol. 2025 Feb 12.

Leveraging prior knowledge to infer gene regulatory networks from single-cell RNA-sequencing data.

Marco Stock, Corinna Losert, Matteo Zambon, Niclas Popp, Gabriele Lubatti, Eva Hörmanseder, Matthias Heinig, Antonio Scialdone.

  Many studies have used single-cell RNA sequencing (scRNA-seq) to infer gene regulatory networks (GRNs), which are crucial for understanding complex cellular regulation. However, the inherent noise and sparsity of scRNA-seq data present significant challenges to accurate GRN inference. This review explores one promising approach that has been proposed to address these challenges: integrating prior knowledge into the inference process to enhance the reliability of the inferred networks. We categorize common types of prior knowledge, such as experimental data and curated databases, and discuss methods for representing priors, particularly through graph structures. In addition, we classify recent GRN inference algorithms based on their ability to incorporate these priors and assess their performance in different contexts. Finally, we propose a standardized benchmarking framework to evaluate algorithms more fairly, ensuring biologically meaningful comparisons. This review provides guidance for researchers selecting GRN inference methods and offers insights for developers looking to improve current approaches and foster innovation in the field.

Keywords:  Gene Regulatory Network Inference; Graph Learning; Prior Knowledge; Single-cell Multiomics; Single-cell Transcriptomics

DOI:  https://doi.org/10.1038/s44320-025-00088-3
Nat Commun. 2025 Feb 10. 16(1): 1501

Mitofusin 2 displays fusion-independent roles in proteostasis surveillance.

Mariana Joaquim, Selver Altin, Maria-Bianca Bulimaga, Tânia Simões, Hendrik Nolte, Verian Bader, Camilla Aurora Franchino, Solenn Plouzennec, Karolina Szczepanowska, Elena Marchesan, Kay Hofmann, Marcus Krüger, Elena Ziviani, Aleksandra Trifunovic, Arnaud Chevrollier, Konstanze F Winklhofer, Elisa Motori, Margarete Odenthal, Mafalda Escobar-Henriques.

Mitochondria are essential organelles and their functional state dictates cellular proteostasis. However, little is known about the molecular gatekeepers involved, especially in absence of external stress. Here we identify a role of MFN2 in quality control independent of its function in organellar shape remodeling. MFN2 ablation alters the cellular proteome, marked for example by decreased levels of the import machinery and accumulation of the kinase PINK1. Moreover, MFN2 interacts with the proteasome and cytosolic chaperones, thereby preventing aggregation of newly translated proteins. Similarly to MFN2-KO cells, patient fibroblasts with MFN2-disease variants recapitulate excessive protein aggregation defects. Restoring MFN2 levels re-establishes proteostasis in MFN2-KO cells and rescues fusion defects of MFN1-KO cells. In contrast, MFN1 loss or mitochondrial shape alterations do not alter protein aggregation, consistent with a fusion-independent role of MFN2 in cellular homeostasis. In sum, our findings open new possibilities for therapeutic strategies by modulation of MFN2 levels.

DOI: https://doi.org/10.1038/s41467-025-56673-5
Nat Commun. 2025 Feb 12. 16(1): 1570

Temporal phosphoproteomics reveals circuitry of phased propagation in insulin signaling.

Michael Turewicz, Christine Skagen, Sonja Hartwig, Stephan Majda, Kristina Thedinga, Ralf Herwig, Christian Binsch, Delsi Altenhofen, D Margriet Ouwens, Pia Marlene Förster, Thorsten Wachtmeister, Karl Köhrer, Torben Stermann, Alexandra Chadt, Stefan Lehr, Tobias Marschall, G Hege Thoresen, Hadi Al-Hasani.

Insulin is a pleiotropic hormone that elicits its metabolic and mitogenic actions through numerous rapid and reversible protein phosphorylations. The temporal regulation of insulin's intracellular signaling cascade is highly complex and insufficiently understood. We conduct a time-resolved analysis of the global insulin-regulated phosphoproteome of differentiated human primary myotubes derived from satellite cells of healthy donors using high-resolution mass spectrometry. Identification and tracking of ~13,000 phosphopeptides over time reveal a highly complex and coordinated network of transient phosphorylation and dephosphorylation events that can be allocated to time-phased regulation of distinct and non-overlapping subcellular pathways. Advanced network analysis combining protein-protein-interaction (PPI) resources and investigation of donor variability in relative phosphosite occupancy over time identifies novel putative candidates in non-canonical insulin signaling and key regulatory nodes that are likely essential for signal propagation. Lastly, we find that insulin-regulated phosphorylation of the pre-catalytic spliceosome complex is associated with acute alternative splicing events in the transcriptome of human skeletal muscle. Our findings highlight the temporal relevance of protein phosphorylations and suggest that synchronized contributions of multiple signaling pathways form part of the circuitry for propagating information to insulin effector sites.

DOI: https://doi.org/10.1038/s41467-025-56335-6
J Proteome Res. 2025 Feb 10.

ProteoArk: A One-Pot Proteomics Data Analysis and Visualization Tool for Biologists.

Mahammad Nisar, Sreelakshmi Pathappillil Soman, Sourav Sreelan, Levin John, Sneha M Pinto, Richard Kumaran Kandasamy, Yashwanth Subbannayya, Thottethodi Subrahmanya Keshava Prasad, Saptami Kanekar, Rajesh Raju, Rex Devasahayam Arokia Balaya.

  ProteoArk is a web-based tool that offers a range of computational pipelines for comprehensive analysis and visualization of mass spectrometry-based proteomics data. The application comprises four primary sections designed to address various aspects of mass spectrometry data analysis in a single platform, including label-free and labeled samples (SILAC/iTRAQ/TMT), differential expression analysis, and data visualization. ProteoArk supports postprocessing of Proteome Discoverer, MaxQuant, and MSFragger search results. The tool also includes functional enrichment analyses such as gene ontology, protein-protein interactions, pathway analysis, and differential expression analysis, which incorporate various statistical tests. By streamlining workflows and developing user-friendly interfaces, we created a robust and accessible solution for users with basic bioinformatics skills in proteomic data analysis. Users can easily create manuscript-ready figures with a single click, including principal component analysis, heatmaps (K-means and hierarchical), MA plots, volcano plots, and circular bar plots. ProteoArk is developed using the Django framework and is freely available for users [https://ciods.in/proteoark/]. Users can also download and run the standalone version of ProteoArk using Docker as described in the instructions [https://ciods.in/proteoark/dockerpage]. The application code, input data, and documentation are available online at https://github.com/ArokiaRex/proteoark. A tutorial video is available on YouTube: https://www.youtube.com/watch?v=WFMKAZ9Slq4&ab_channel=RexD.A.B.

Keywords:  bioinformatics tools; data analysis; data visualization; mass spectrometry; proteomics; web application

DOI:  https://doi.org/10.1021/acs.jproteome.4c00556
Elife. 2025 Feb 12. pii: RP98110. [Epub ahead of print]13

A molecular proximity sensor based on an engineered, dual-component guide RNA.

Junhong Choi, Wei Chen, Hanna Liao, Xiaoyi Li, Jay Shendure.

  One of the goals of synthetic biology is to enable the design of arbitrary molecular circuits with programmable inputs and outputs. Such circuits bridge the properties of electronic and natural circuits, processing information in a predictable manner within living cells. Genome editing is a potentially powerful component of synthetic molecular circuits, whether for modulating the expression of a target gene or for stably recording information to genomic DNA. However, programming molecular events such as protein-protein interactions or induced proximity as triggers for genome editing remains challenging. Here, we demonstrate a strategy termed 'P3 editing', which links protein-protein proximity to the formation of a functional CRISPR-Cas9 dual-component guide RNA. By engineering the crRNA:tracrRNA interaction, we demonstrate that various known protein-protein interactions, as well as the chemically induced dimerization of protein domains, can be used to activate prime editing or base editing in human cells. Additionally, we explore how P3 editing can incorporate outputs from ADAR-based RNA sensors, potentially allowing specific RNAs to induce specific genome edits within a larger circuit. Our strategy enhances the controllability of CRISPR-based genome editing, facilitating its use in synthetic molecular circuits deployed in living cells.

Keywords:  CRISPR-Cas; genetics; genome editing; genomics; human; molecular recording; protein-protein interaction; synthetic biology

DOI:  https://doi.org/10.7554/eLife.98110
ACS Chem Biol. 2025 Feb 11.

Workflow for E3 Ligase Ligand Validation for PROTAC Development.

Nebojša Miletić, Janik Weckesser, Thorsten Mosler, Rajeshwari Rathore, Marina E Hoffmann, Paul Gehrtz, Sarah Schlesiger, Ingo V Hartung, Nicola Berner, Stephanie Wilhelm, Juliane Müller, Bikash Adhikari, Václav Němec, Saran Aswathaman Sivashanmugam, Lewis Elson, Hanna Holzmann, Martin P Schwalm, Lasse Hoffmann, Kamal Rayees Abdul Azeez, Susanne Müller, Bernhard Kuster, Elmar Wolf, Ivan Đikić, Stefan Knapp.

Proteolysis targeting chimeras (PROTACs) have gained considerable attention as a new modality in drug discovery. The development of PROTACs has been mainly focused on using CRBN (Cereblon) and VHL (Von Hippel-Lindau ligase) E3 ligase ligands. However, the considerable size of the human E3 ligase family, newly developed E3 ligase ligands, and the favorable druggability of some E3 ligase families hold the promise that novel degraders with unique pharmacological properties will be designed in the future using this large E3 ligase space. Here, we developed a workflow aiming to improve and streamline the evaluation of E3 ligase ligand efficiency for PROTAC development and the assessment of the corresponding "degradable" target space using broad-spectrum kinase inhibitors and the well-established VHL ligand VH032 as a validation system. Our study revealed VH032 linker attachment points that are highly efficient for kinase degradation as well as some of the pitfalls when using protein degradation as a readout. For instance, cytotoxicity was identified as a major mechanism leading to PROTAC- and VHL-independent kinase degradation. The combination of E3 ligase ligand negative controls, competition by kinase parent compounds, and neddylation and proteasome inhibitors was essential to distinguish between VHL-dependent and -independent kinase degradation events. We share here the findings and limitations of our study and hope that this study will provide guidance for future evaluations of new E3 ligase ligand systems for degrader development.

DOI: https://doi.org/10.1021/acschembio.4c00812
Cell Rep. 2025 Feb 12. pii: S2211-1247(25)00052-X. [Epub ahead of print]44(2): 115281

Inositol polyphosphate multikinase regulates Th1 and Th17 cell differentiation by controlling Akt-mTOR signaling.

Chae Min Yuk, Sehoon Hong, Dongeon Kim, Mingyo Kim, Hyun-Woo Jeong, Seung Ju Park, Hyungyu Min, Wooseob Kim, Jongbu Lim, Hyo Dam Kim, Sang-Gyu Kim, Rho Hyun Seong, Seyun Kim, Seung-Hyo Lee.

  Activated proinflammatory T helper (Th) cells, including Th1 and Th17 cells, drive immune responses against pathogens and contribute to autoimmune diseases. We show that the expression of inositol polyphosphate multikinase (IPMK), an enzyme essential for inositol phosphate metabolism, is highly induced in Th1 and Th17 subsets. Deletion of IPMK in CD4+ T cells leads to diminished Th1- and Th17-mediated responses, reducing resistance to Leishmania major and attenuating experimental autoimmune encephalomyelitis. IPMK-deficient CD4+ T cells show impaired activation and Th17 differentiation, linked to the decreased activation of Akt, mTOR, and STAT3. Mechanistically, IPMK functions as a phosphatidylinositol 3-kinase to regulate phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) production, promoting T cell activation and effector functions. In IPMK-deficient CD4+ T cells, T cell receptor-stimulated PtdIns(3,4,5)P3 generation is abolished by wortmannin, suggesting IPMK acts in a wortmannin-sensitive manner. These findings establish IPMK as a critical regulator of Th1 and Th17 differentiation, underscoring its role in maintaining immune homeostasis.

Keywords:  CD4(+) T cell differentiation; CD4(+) T cells; CP: Immunology; CP: Metabolism; EAE; IPMK; Leishmania major infection; PIP3-Akt-mTOR signaling; STAT3 signaling; Th1 cells; Th17 cells; experimental autoimmune encephalomyelitis; inositol polyphosphate multikinase

DOI:  https://doi.org/10.1016/j.celrep.2025.115281