bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2025–05–04
38 papers selected by
Ralitsa Radostinova Madsen, MRC-PPU



  1. Proc Natl Acad Sci U S A. 2025 May 06. 122(18): e2416516122
      Constructing single-cell atlases requires preserving differences attributable to biological variables, such as cell types, tissue origins, and disease states, while eliminating batch effects. However, existing methods are inadequate in explicitly modeling these biological variables. Here, we introduce SIGNAL, a general framework that leverages biological variables to disentangle biological and technical effects, thereby linking these metadata to data integration. SIGNAL employs a variant of principal component analysis to align multiple batches, enabling the integration of 1 million cells in approximately 2 min. SIGNAL, despite its computational simplicity, surpasses state-of-the-art methods across multiple integration scenarios: 1) heterogeneous datasets, 2) cross-species datasets, 3) simulated datasets, 4) integration on low-quality cell annotations, and 5) reference-based integration. Furthermore, we demonstrate that SIGNAL accurately transfers knowledge from reference to query datasets. Notably, we propose a self-adjustment strategy to restore annotated cell labels potentially distorted during integration. Finally, we apply SIGNAL to multiple large-scale atlases, including a human heart cell atlas containing 2.7 million cells, identifying tissue- and developmental stage-specific subtypes, as well as condition-specific cell states. This underscores SIGNAL's exceptional capability in multiscale analysis.
    Keywords:  data integration; knowledge transfer; principal component analysis; single-cell data; technical variation
    DOI:  https://doi.org/10.1073/pnas.2416516122
  2. Development. 2025 Apr 15. pii: dev204493. [Epub ahead of print]152(8):
      The Drosophila pseudokinase Tribbles (Trbl) shares conserved functions with human TRIB3 to bind and inhibit Akt phosphorylation-activation by the Insulin Receptor (InR) to reduce insulin responses; consistent with this, increased levels of human TRIB3 are linked to type 2 diabetes. Here, we show that in fat body cells of well-fed Drosophila larvae, Trbl expression is low and predominantly in the nucleus while fasting or genetic reduction of insulin signaling resulted in increased Trbl expression and Trbl protein translocation to the plasma membrane. An E/G mutation in the Trbl pseudokinase kinase activation loop dominantly interfered with Trbl function leading to increased Akt activity, increased stability of Trbl substrates, including Trbl itself, and aberrant redistribution of Trbl multimers to the membrane. Several strategies designed to increase Akt activity were sufficient to translocate Trbl to the membrane, consistent with the notion that subcellular trafficking of Trbl to the fat body cell membrane acts a rheostat to reduce the strength of Akt-mediated insulin responses, counter to the InR, which has been shown to redistribute away from the membrane to modulate insulin signaling.
    Keywords:  Growth; Insulin signaling; Pseudokinase; Trib protein family
    DOI:  https://doi.org/10.1242/dev.204493
  3. Commun Biol. 2025 Apr 26. 8(1): 666
      Understanding cellular diversity and disease mechanisms requires a global analysis of proteins and their modifications. While next-generation sequencing has advanced our understanding of cellular heterogeneity, it fails to capture downstream signalling networks. Ultrasensitive mass spectrometry-based proteomics enables unbiased protein-level analysis of low cell numbers, down to single cells. However, phosphoproteomics remains limited to high-input samples due to sample losses and poor reaction efficiencies associated with processing low cell numbers. Isobaric stable isotope labelling is a promising approach for reproducible and accurate quantification of low abundant phosphopeptides. Here, we introduce SPARCE (Streamlined Phosphoproteomic Analysis of Rare CElls) for multiplexed phosphoproteomic analysis of low cell numbers. SPARCE integrates cell isolation, water-based lysis, on-tip TMT labelling, and phosphopeptide enrichment. SPARCE outperforms traditional methods by enhancing labelling efficiency and phosphoproteome coverage. To demonstrate the utility of SPARCE, we analysed four patient-derived glioblastoma stem cell lines, reliably quantifying phosphosite changes from 1000 FACS-sorted cells. This workflow expands the possibilities for signalling analysis of rare cell populations.
    DOI:  https://doi.org/10.1038/s42003-025-08068-x
  4. Semin Cell Dev Biol. 2025 Apr 24. pii: S1084-9521(25)00025-4. [Epub ahead of print]171 103615
      Apoptosis is now recognized as a highly dynamic process that involves the release of a large set of signaling molecules that convey information to cells neighboring an apoptotic site. Recent studies in epithelial systems have discovered that apoptotic cells trigger waves of pulses of mitogen-activated protein kinase (MAPK) / extracellular signal-regulated kinase (ERK) pathway activity in their neighbors. At the single-cell level, the ERK pulses emerge from the MAPK pathway's excitable network properties, such as ultrasensitivity and adaptation. At the cell population level, apoptosis-induced ERK waves (AiEWs) emerge from propagation of ERK pulses across cells via a mechanism that involves mechanical inputs and paracrine signaling. AiEWs enable cell populations to dynamically coordinate fate decision signaling during tissue homeostasis and development. This spatio-temporal signaling mechanism can be hijacked by cancer cells to induce drug-tolerant persister states when apoptosis is triggered by cytotoxic or targeted therapies, undermining treatment efficacy. In this review, we summarize our current understanding of AiEWs, including their initiation, propagation, and coordination of fate decision signaling within a population. We discuss how the relatively simple properties of single cells, and their interactions within a collective coordinate these dynamic signaling patterns. We highlight their implication in resistance to cancer therapy and explore potential strategies to target these waves to re-sensitize cancer cells. Finally, we discuss emerging technologies and future directions to expand the study of this biological phenomenon.
    Keywords:  Apoptosis; Cancer therapy resistance; Cell fate decisions; Cellular communication; Collective behavior; Drug-tolerant persister state; ERK dynamics; ERK waves; Emergent properties; Epithelial monolayer; Epithelial physiology; Fluorescent biosensors; MAPK pathway; Mechanotransduction; Quantitative cell biology; Signal transduction; Single-cell dynamics; Spatiotemporal signaling; Tissue homeostasis; Tissue selforganization
    DOI:  https://doi.org/10.1016/j.semcdb.2025.103615
  5. EMBO Rep. 2025 Apr 30.
      Ependymal cells are multiciliated glial cells lining the ventricles of the mammalian brain. Their differentiation from progenitor cells involves cell enlargement and progresses through centriole amplification phases and ciliogenesis. These phases are accompanied by the sharp up-regulation of mTOR Complex 1 activity (mTORC1), a master regulator of macromolecule biosynthesis and cell growth, whose function in ependymal cell differentiation is unknown. We demonstrate that mTORC1 inhibition by rapamycin preserves the progenitor pool by reinforcing quiescence and preventing alternative cell cycle progression for centriole amplification. Overexpressing E2F4 and MCIDAS circumvents mTORC1-regulated processes, enabling centriole amplification despite rapamycin, and enhancing mTORC1 activity through positive feedback. Acute rapamycin treatment in multicentriolar cells during the late phases of differentiation causes centriole regrouping, indicating a direct role of mTORC1 in centriole dynamics. By phosphoproteomic and phosphomutant analysis, we reveal that the mTORC1-mediated phosphorylation of GAS2L1, a centrosomal protein that links actin and microtubule cytoskeletons, participates in centriole disengagement. This multilayered and sequential control of ependymal development by mTORC1, from the progenitor pool to centriolar function, has implications for pathophysiological conditions like aging and hydrocephalus-prone genetic diseases.
    Keywords:  Cell Cycle; Ciliogenesis; Cytoskeleton; Differentiation; mTOR
    DOI:  https://doi.org/10.1038/s44319-025-00460-2
  6. Science. 2025 May;388(6746): eadr5499
      The ability to study proteins in their native cellular context is crucial to our understanding of biology. In this work, we report a technology for intracellular protein editing, drawing from split intein-mediated protein splicing, genetic code expansion, and endogenous protein tagging. This approach enables us to rapidly and site-specifically install residues and chemical handles into a protein. We demonstrate the power of this platform to edit cellular proteins, inserting epitopes, protein-specific sequences, and noncanonical amino acids. Notably, we use an endogenous tagging approach to apply our protein editing technology to endogenous proteins with minimal perturbation. We anticipate that the protein editing technology presented in this work will be applied to a diverse set of problems and phenomena in live mammalian cells.
    DOI:  https://doi.org/10.1126/science.adr5499
  7. Nucleic Acids Res. 2025 May 01. pii: gkaf365. [Epub ahead of print]
      Since its introduction in 2003, Cytoscape has been a de facto standard for visualizing and analyzing biological networks. We now introduce Cytoscape Web (https://web.cytoscape.org), an online implementation that captures the interface and key visualization functionality of the desktop while providing integration with web tools and databases. Cytoscape Web enhances accessibility, simplifying collaboration through online data sharing. It integrates with Cytoscape desktop via the CX2 network exchange format and with the Network Data Exchange for storing and sharing networks. The platform supports extensibility through an App framework for UI components and Service Apps for algorithm integration, fostering community-driven development of new analysis tools. Overall, Cytoscape Web enhances network biology by providing a versatile, accessible, and collaborative online platform that adapts to evolving computational challenges, laying a foundation for future incorporation of advanced network analysis capabilities by the community.
    DOI:  https://doi.org/10.1093/nar/gkaf365
  8. bioRxiv. 2025 Apr 26. pii: 2025.04.23.650349. [Epub ahead of print]
      Cytosolic arginine sensor for mTORC1 Subunit 1 (CASTOR1) functions as a key regulator of mechanistic target of rapamycin complex 1 (mTORC1) signaling. Despite its frequent dysregulation in cancers via mechanisms such as KSHV microRNA-mediated inhibition or AKT-driven phosphorylation and degradation, the impact of CASTOR1 loss on tumor initiation and progression remains poorly understood. Here, we identify CASTOR1 as a critical tumor suppressor in non-small cell lung cancer (NSCLC) by demonstrating that its genetic ablation amplifies tumorigenesis in a KRAS -driven genetically engineered mouse model (GEMM;LSL- KRAS G12D ). CASTOR1 deficiency markedly enhances lung tumor incidence, accelerates tumor progression, and increases proliferative indices in KRAS G12D -driven tumors ( KRAS G12D ; C1 KO ) compared to CASTOR1 wild type (WT) tumors ( KRAS G12D ; C1 WT ). Advanced-stage tumors exhibit elevated phosphorylated CASTOR1 (pCASTOR1) and reduced total CASTOR1 levels, suggesting active degradation during tumorigenesis. Mechanistically, CASTOR1 loss amplifies mTORC1 signaling, as evidenced by heightened phosphorylation of downstream effectors 4EBP1 and S6, while also augmenting AKT and ERK activation, uncovering a crosstalk between the PI3K/AKT/mTORC1 and KRAS/ERK pathways. Furthermore, CASTOR1 ablation induces genome instability, which may contribute to enhanced tumor incidence and progression. Importantly, CASTOR1 deficiency confers resistance to KRAS G12D -specific inhibitors, while over half of KRAS G12D ; C1 WT tumors also display resistance. Organoids derived from KRAS G12D ; C1 KO and KRAS G12D ; C1 WT tumors reveal a correlation between KRAS inhibitor resistance and hyperactivation of mTORC1, with mTORC1 and PI3K inhibitors sensitizing resistant tumors to KRAS G12D -targeted therapies. These findings position CASTOR1 as a novel tumor suppressor that modulates mTORC1 and KRAS signaling to constrain NSCLC progression. Our study further highlights the therapeutic potential of combining mTORC1 or ERK inhibitors with KRAS-targeted therapies for NSCLC characterized by hyperactive KRAS signaling and impaired CASTOR1 activity.
    Highlights: CASTOR1 functions as a tumor suppressor in NSCLC by limiting KRAS -driven tumor initiation and progression. CASTOR1 is frequently lost or inactivated in wild-type tumors during tumor progression, contributing to advanced-stage malignancies.CASTOR1 deficiency amplifies mTORC1 signaling and enhances PI3K/AKT and KRAS/ERK crosstalk, driving tumorigenesis and resistance to KRAS-specific inhibitors. Combining mTORC1 or PI3K inhibitors with KRAS-targeted therapies effectively overcomes resistance in KRAS -driven NSCLC.
    DOI:  https://doi.org/10.1101/2025.04.23.650349
  9. bioRxiv. 2025 Apr 09. pii: 2025.04.04.647273. [Epub ahead of print]
      EGFR is a proto-oncogene that is mutationally activated in a variety of cancers. Small molecule inhibitors targeting EGFR can be effective in slowing the progression of disease, and in some settings these drugs even cause dramatic tumor regression. However, responses to EGFR inhibitors are rarely durable, and the mechanisms contributing to response variation remain unclear. In particular, several distinct mechanisms have been proposed for how EGFR inhibition activates cell death, and a consensus has yet to emerge. In this study, we use functional genomics with specialized analyses to infer how genetic perturbations effect the drug-induced death rate. Our data clarify that inhibition of PI3K signaling drives the lethality of EGFR inhibition. Inhibition of other pathways downstream of EGFR, including the RAS-MAPK pathway, promote growth suppression, but not the lethal effects of EGFR inhibitors. Taken together, our study reveals the first "reference map" for the genome-wide genetic dependencies of lethality for EGFR inhibitors.
    DOI:  https://doi.org/10.1101/2025.04.04.647273
  10. Cell Stem Cell. 2025 May 01. pii: S1934-5909(25)00140-7. [Epub ahead of print]32(5): 673-675
      Metabolic regulation of embryonic development is increasingly recognized. Villaronga-Luque et al.1 and Stopornwongkul et al.2 show that metabolic activity influences gastruloid formation from mouse embryonic stem cells, revealing that the balance between glycolysis and oxidative phosphorylation regulates cell fate decisions during gastruloid self-organization.
    DOI:  https://doi.org/10.1016/j.stem.2025.04.005
  11. Res Sq. 2025 Apr 25. pii: rs.3.rs-6234375. [Epub ahead of print]
      Proteinuria is a widely utilized surrogate marker in clinical practice for its predictive and prognostic value. The mechanistic link between proteinuria and progression remains elusive. Proximal tubule epithelial cells(PTEC) retrieve albumin in the glomerular filtrate via receptor mediated endocytosis facilitated by megalin-cubilin complex. We reported that cell-survival protein, Akt phosphorylates cargo binding endocytic adaptor protein to megalin, disabled-2(Dab2). We hypothesize that downregulation of Akt signaling as a result of overwhelmed endocytic machinery in albumin overload is linked to PTEC apoptosis in proteinuric states. We show that cell culture and animal model of albumin overload inhibited phosphorylation of Akt in association with apoptosis in PTEC. Chemical inhibition and overexpression of Akt by constitutively active Akt plasmid exacerbated and alleviated apoptosis respectively in response to albumin overload in PTEC. Mouse with targeted inhibition of Akt1 and Akt2 in PTEC (Akt1/2lox/loxSGLT2cre) displayed perturbed albumin endocytosis at baseline. Albumin overload in Akt1/2 lox/lox SGLT2cre mouse led to dephosphorylation and translocation downstream Akt target, Forkhead box O-1 (Foxo1) to nuclei driving transcriptional activation of proapoptotic BIM followed by translocation of proapoptotic Bax and BIM to mitochondria and cytochrome-c to cytosol. In an effort to investigate the role of Akt in progression, we examined kidney biopsy specimens of patients with focal segmental glomerulosclerosis (FSGS) and minimal change disease. Kidney biopsies of patients with FSGS exhibited decreased pSer473-Akt expression in PTEC early in the course of disease, preceding progression to end stage kidney disease. We conclude that downstream dephosphorylation of Foxo and transcriptional activation of BIM and subsequent mitochondrial injury drives apoptosis following Akt downregulation in PTEC albeit inhibition of albumin endocytosis in proteinuric states.
    DOI:  https://doi.org/10.21203/rs.3.rs-6234375/v1
  12. ArXiv. 2025 Apr 10. pii: arXiv:2504.08096v1. [Epub ahead of print]
      Cellular development follows a stochastic yet rule-governed trajectory, though the underlying principles remain elusive. Here, we propose that cellular development follows paths of least action, aligning with foundational physical laws that govern dynamic systems across nature. We introduce a computational framework that takes advantage of the deep connection between the principle of least action and maximum entropy to model developmental processes using Transformers architecture. This approach enables precise quantification of entropy production, information flow curvature, and local irreversibility for developmental asymmetry in single-cell RNA sequence data. Within this unified framework, we provide interpretable metrics: entropy to capture exploration-exploitation trade-offs, curvature to assess plasticity-elasticity dynamics, and entropy production to characterize dedifferentiation and transdifferentiation. We validate our method across both single-cell and embryonic development datasets, demonstrating its ability to reveal hidden thermodynamic and informational constraints shaping cellular fate decisions.
  13. Cell Syst. 2025 Apr 30. pii: S2405-4712(25)00098-5. [Epub ahead of print] 101265
      To investigate how cellular variations arise across spatiotemporal scales in a population of identical healthy cells, we performed a data-driven analysis of nuclear growth variations in hiPS cell colonies as a model system. We generated a 3D timelapse dataset of thousands of nuclei over multiple days and developed open-source tools for image and data analysis and feature-based timelapse data exploration. Together, these data, tools, and workflows comprise a framework for systematic quantitative analysis of dynamics at individual and population levels, and the analysis further highlights important aspects to consider when interpreting timelapse data. We found that individual nuclear volume growth trajectories arise from short-timescale variations attributable to their spatiotemporal context within the colony. We identified a time-invariant volume compensation relationship between nuclear growth duration and starting volume across the population. Notably, we discovered that inheritance plays a crucial role in determining these two key nuclear growth features while other growth features are determined by their spatiotemporal context and are not inherited.
    Keywords:  induced pluripotent stem cells; inheritance of cellular organization; nuclear growth dynamics; timelapse data analysis
    DOI:  https://doi.org/10.1016/j.cels.2025.101265
  14. bioRxiv. 2025 Apr 08. pii: 2025.04.03.647044. [Epub ahead of print]
      From bacteria to humans, most organisms showcase inherent 24-hour circadian rhythms, best exemplified by the sleep-wake cycle. These rhythms are remarkably widespread, governing hormonal, metabolic, physiological, and behavioral oscillations, and are driven by "molecular clocks" that orchestrate the rhythmic expression of thousands of genes throughout the body. Here, we generate single-cell RNA and ATAC multiomic data to simultaneously characterize gene expression and chromatin accessibility of ∼33,000 mouse liver cells across the 24-hour day. Our study yields several key insights, including: (i) detecting circadian rhythmicity in both discretized liver cell types and transient sub-lobule cell states, capturing space-time RNA and ATAC profiles in a cell-type- and cell-state-specific manner; (ii) delving beyond mean cyclic patterns to characterize distributions, accounting for gene expression stochasticity due to transcriptional bursting; (iii) interrogating multimodal circadian rhythmicity, encompassing RNAs, DNA regulatory elements, and transcription factors (TFs), while examining priming and lagging effects across modalities; and (iv) inferring spatiotemporal gene regulatory networks involving target genes, TFs, and cis-regulatory elements that controls circadian rhythmicity and liver physiology. Our findings apply to existing single-cell data of mouse and Drosophila brains and are further validated by time-series single molecule fluorescence in situ hybridization, as well as vast amounts of existing and orthogonal high-throughput data from chromatin immunoprecipitation followed by sequencing, capture Hi-C, and TF knockout experiments. Altogether, our study constructs a comprehensive map of the time-series transcriptomic and epigenomic landscapes that elucidate the function and mechanism of the liver peripheral clocks.
    DOI:  https://doi.org/10.1101/2025.04.03.647044
  15. Nat Commun. 2025 Apr 30. 16(1): 4071
      Spatial omics data provide rich molecular and structural information on tissues. Their analysis provides insights into local heterogeneity of tissues and holds promise to improve patient stratification by associating clinical observations with refined tissue representations. We introduce Kasumi, a method for identifying spatially localized neighborhood patterns of intra- and intercellular relationships that are persistent across samples and conditions. The tissue representation based on these patterns can facilitate translational tasks, as we show for stratification of cancer patients for disease progression and response to treatment using data from different experimental platforms. On these tasks, Kasumi outperforms related approaches and offers explanations of spatial coordination and relationships at the cell-type or marker level. We show that persistent patterns comprise regions of different sizes, and that non-abundant, localized relationships in the tissue are strongly associated with unfavorable outcomes.
    DOI:  https://doi.org/10.1038/s41467-025-59448-0
  16. Biology (Basel). 2025 Apr 05. pii: 374. [Epub ahead of print]14(4):
      The ERK pathway is an important biochemical cascade and acts as a master regulator of myriad cell processes including cell proliferation, differentiation, and survival. Early biochemical work established that the timing of ERK phosphorylation was an important determinant of PC12 cell fate, with extended phosphorylation (with nerve growth factor treatment) linked to differentiation but rapid on-off ERK phosphorylation kinetics (with epidermal growth factor treatment) linked to cell proliferation. Recent work from several laboratories has revealed that periodic forcing the phosphorylation of ERK with growth factors, light (optogenetics) or electronically can switch cell fate from proliferative to differentiated depending on type of stimulus (amplitude and frequency). Here, we take an ERK model and analyze it from the frequency domain perspective. The key is the transfer function, which provides a compact description of input (growth factor)-output (ERK activation) behavior over a range of input frequencies, allowing an understanding of system dynamics in terms of amplitude modulations, phase shifts, and signaling bandwidths. Our analysis of transfer functions indicates that, at normal receptor levels, the ERK pathway acts as a negative feedback amplifier to growth factor fluctuations, amplifying them at low receptor occupancy but suppressing them at high receptor occupancy. The frequency dependence is best described as a resonant low pass filter, which selectively filters out high frequency input oscillations. We use the transfer function to predict how different growth factor input dynamics shape ERK activation.
    Keywords:  EGF; ERK pathway; NGF; PC12; frequency domain; transfer function
    DOI:  https://doi.org/10.3390/biology14040374
  17. J Biol Chem. 2025 Apr 24. pii: S0021-9258(25)00394-1. [Epub ahead of print] 108545
      Small GTPases play crucial roles in cellular signaling pathways, with their activation states tightly regulated between GDP-bound inactive and GTP-bound active forms. Dysregulation of these nucleotide-binding states, such as in oncogenic RAS, is implicated in diseases like cancer. Accurately quantifying these states in cells is thus crucial for deciphering their functional roles and regulatory mechanisms. However, current methods do not fully meet the necessary sensitivity and versatility, limiting their effectiveness in small GTPase analysis. Here, we present a highly sensitive HPLC-based assay with fluorescence detection (Fluor-HPLC), enabling precise quantification of guanine nucleotide-binding states in small GTPases. Applying this technique, we successfully quantified the guanine nucleotide-binding states of small GTPases at their endogenous expression levels. We demonstrated the utility of Fluor-HPLC by elucidating RHEB and HRAS activation in response to extracellular stimuli. Furthermore, integration of Fluor-HPLC with syngeneic mouse models provided insights into KRAS activation dynamics in tumor tissues and evaluated the effectiveness of targeted therapeutics. Overall, this versatile method paves the way for investigating activation states and regulatory mechanisms of various small GTPases, potentially accelerating our understanding of their roles in cellular processes and disease pathogenesis.
    Keywords:  HRAS; KRAS; RHEB; anticancer drug; high‐performance liquid chromatography (HPLC); small GTPase
    DOI:  https://doi.org/10.1016/j.jbc.2025.108545
  18. Nature. 2025 May;641(8061): 270-271
      
    Keywords:  Authorship; Communication; Microscopy; Research data; Technology
    DOI:  https://doi.org/10.1038/d41586-025-01299-2
  19. Proteomics. 2025 Apr 26. e202400417
      The accurate construction of computational models in systems biology heavily relies on the availability of quantitative proteomics data, specifically, absolute protein abundances. However, the complex nature of proteomics data analysis necessitates specialised expertise, making the integration of this data into models challenging. Therefore, the development of software tools that ease the analysis of proteomics data and bridge between disciplines is crucial for advancing the field of systems biology. We developed an open access Python-based software tool available either as downloadable library or as web-based graphical user interface (GUI). The pipeline simplifies the extraction and calculation of protein abundances from unprocessed proteomics data, accommodating a range of experimental approaches based on label-free quantification. Our tool was conceived as a versatile and robust pipeline designed to ease and simplify data analysis, thereby improving reproducibility between researchers and institutions. Moreover, the robust modular structure of Alpaca allows its integration with other software tools.
    Keywords:  Python; absolute proteome quantification; data mining; open source; protein abundances; proteomics; proteomics analysis
    DOI:  https://doi.org/10.1002/pmic.202400417
  20. Cancer Discov. 2025 Apr 28. OF1-OF18
       SIGNIFICANCE: RAS inhibition in multiple tumor types reveals the difference between G12 mutants and Q61 mutants in their cooperation with upstream regulators and downstream effectors to promote oncogenic signaling. Our findings provide the rationale for combinatorial approaches and contribute to explaining the nonuniform distribution of RAS mutations, de novo and at resistance.
    DOI:  https://doi.org/10.1158/2159-8290.CD-24-0614
  21. bioRxiv. 2025 Apr 08. pii: 2025.04.02.646782. [Epub ahead of print]
       Background: Multiplexed tissue imaging enables the simultaneous detection of dozens of proteins at single-cell resolution, providing unprecedented insights into tissue organization and disease microenvironments. However, the resulting high-dimensional, gigabyte-scale datasets pose significant computational and methodological challenges. Existing analytical workflows, often fragmented between bespoke scripts and static visualizations, lack the scalability and user-friendly interfaces required for efficient, reproducible analysis. To overcome these limitations, we developed SPAC (analysis of SPAtial single-Cell datasets), a scalable, web-based ecosystem that integrates modular pipelines, high-performance computing (HPC) connectivity, and interactive visualization to democratize end-to-end single-cell spatial analysis applied to cellular positional data and protein expression levels.
    Results: SPAC is built on a modular, layered architecture that leverages community-based and newly developed tools for single-cell and spatial proteomics analysis. A specialized Python package extends these functionalities with custom analysis routines and established software engineering practices. An Interactive Analysis Layer provides web-hosted pipelines for configuring and executing complex workflows, and scalability enhancements that support distributed or parallel execution on GPU-enabled clusters. A Real-Time Visualization Layer delivers dynamic dashboards for immediate data exploration and sharing. As a showcase of its capabilities, SPAC was applied to a 4T1 breast cancer model, analyzing a multiplex imaging dataset comprising 2.6 million cells. GPU acceleration reduced unsupervised clustering runtimes from several hours to under ten minutes, and real-time visualization enabled detailed spatial characterization of tumor subregions.
    Conclusions: SPAC effectively overcomes key challenges in spatial single-cell analysis by streamlining high-throughput data processing and spatial profiling within an accessible and scalable framework. Its robust architecture, interactive interface and ease of access have the potential to accelerate biomedical research and clinical applications by converting complex imaging data into actionable biological and clinical insights.
    DOI:  https://doi.org/10.1101/2025.04.02.646782
  22. Nat Commun. 2025 Apr 25. 16(1): 3919
      Receptor tyrosine kinases (RTKs) play a crucial role in the regulation of intracellular signal transduction, underscoring their significance as targets for drug therapy. Despite the widespread clinical use of kinase inhibitors, the increasing occurrence of off-target effects and drug resistance makes it urgent to explore alternative approaches to modulate RTKs functions. Here, we propose an approach for attenuating cell-surface receptor signaling, termed Aptamer-directed Phosphatase Recruiting Chimeras (Apt-PRCs). The Apt-PRC is composed of an aptamer to recruit phosphatases and a binder to target receptors. As a proof-of-concept, we design and construct Apt-PRCs intended for direct dephosphorylation of tyrosine residues on the receptor targets, i.e., epidermal growth factor receptor and mesenchymal-epithelial transition factor, respectively. The as-developed Apt-PRCs manage to inhibit specifically and efficiently the reception and transmission of phosphorylation signals both in vitro and in vivo. Furthermore, it is discovered that the induced dephosphorylation could enhance the susceptibility to gefitinib in drug-resistant cancer cells and a xenograft mouse model, indicating the potential of Apt-PRCs to overcome drug resistance in cancer. This work offers a versatile methodology to design molecular mediators to modulate receptor phosphorylation so as to regulate the downstream signal transduction and overcome drug resistance.
    DOI:  https://doi.org/10.1038/s41467-025-59098-2
  23. Aging Cell. 2025 Apr 30. e70075
      The differentiation of human pluripotent stem cells (hPSCs) provides access to a wide range of cell types and tissues. However, hPSC-derived lineages typically represent a fetal stage of development, and methods to expedite the transition to an aged identity to improve modeling of late-onset disease are limited. In this study, we introduce RNAge, a transcriptome-based computational platform designed to enable the evaluation of an induced aging or a rejuvenated state. We validated this approach across independent datasets spanning different tissues and species, and show that it can be used to evaluate the effectiveness of existing age-retaining or age-modulating interventions. We also used RNAge to perform an in silico compound screen using the LINCS L1000 dataset. This approach led to the identification and experimental confirmation of several novel compounds capable of inducing aging or rejuvenation in primary fibroblasts or hPSC-derived neurons. Additionally, we observed that applying this novel induced aging strategy to an hPSC model of Alzheimer's disease (AD) accelerated neurodegeneration in a genotype-specific manner. Our study offers a robust method for quantifying age-related manipulations and unveils compounds that significantly broaden the toolkit for age-modifying strategies in hPSC-derived lineages.
    Keywords:  Alzheimer's disease; age score; aging; cortical neurons; disease modeling; human pluripotent stem cells; transcriptional age
    DOI:  https://doi.org/10.1111/acel.70075
  24. Biotechnol J. 2025 Apr;20(4): e70022
      Human induced pluripotent stem cells (iPSCs) are an invaluable endless cell source for generating various therapeutic cells and tissues. However, their differentiation into specific cell lineages, such as definitive endoderm (DE) and pancreatic progenitor (PP), often suffers from poor reproducibility, due partially to their pluripotency. In this work, we investigated the impact of iPSC confluency during cell self-renewal and seeding density on cell metabolic activity, glycolysis to oxidative phosphorylation shift, and differentiation potential toward DE and PP lineages. Our findings demonstrated that cell seeding strategy influences cellular metabolic activity and the robustness of iPSC differentiation. iPSCs maintained at higher seeding density exhibited lower initial oxygen consumption rate (OCR) and metabolic activity. There is an optimal seeding density to ensure sufficient oxygen consumption during differentiation and to yield high expression of SOX17 in the DE lineage and high PDX1/NKX6.1 dual-positive cells in PPs. Interestingly, we found that cell confluency at the time of harvest has less impact on the efficacy of pancreatic lineage formation or metabolic activity. This study sheds light on the interplay between metabolic activity and iPSC lineage specification, offering new insights into the robustness of iPSC self-renewal and differentiation for creating human tissues.
    Keywords:  cell seeding; differentiation; human induced pluripotent stem cells; metabolism; oxygen consumption; pancreatic progenitors
    DOI:  https://doi.org/10.1002/biot.70022
  25. Science. 2025 May;388(6746): eadp2959
      Stem cells are a promising source for cellular therapies across many diseases and tissues. Their inherent ability to differentiate into other cell types has been the focus of investigation over decades. This ability is currently being exploited for therapies using strategies to repair or replace damaged tissues and cells or to alleviate immune rejection. Exploring stem cell function has enabled direct reprogramming approaches, for example, through the production of induced pluripotent stem cells and the generation of tissue-specific stem cells. Understanding stem cell function has emerged as an important strategy for repopulating stem cell pools or generating differentiated cells for therapy. Here, we review general principles of mammalian stem cell biology and cellular reprogramming approaches and their use for current and future therapeutic purposes.
    DOI:  https://doi.org/10.1126/science.adp2959
  26. Nat Chem Biol. 2025 Apr 28.
      Monitoring H2O2 dynamics in conjunction with key biological interactants is critical for elucidating the physiological outcome of cellular redox regulation. Optogenetic hydrogen peroxide sensor with HaloTag with JF635 (oROS-HT635) allows fast and sensitive chemigenetic far-red H2O2 imaging while overcoming drawbacks of existing red fluorescent H2O2 indicators, including oxygen dependency, high pH sensitivity, photoartifacts and intracellular aggregation. The compatibility of oROS-HT635 with blue-green-shifted optical tools allows versatile optogenetic dissection of redox biology. In addition, targeted expression of oROS-HT635 and multiplexed H2O2 imaging enables spatially resolved imaging of H2O2 targeting the plasma membrane and neighboring cells. Here we present multiplexed use cases of oROS-HT635 with other green fluorescence reporters by capturing acute and real-time changes in H2O2 with intracellular redox potential and Ca2+ levels in response to auranofin, an inhibitor of antioxidative enzymes, via dual-color imaging. oROS-HT635 enables detailed insights into intricate intracellular and intercellular H2O2 dynamics, along with their interactants, through spatially resolved, far-red H2O2 imaging in real time.
    DOI:  https://doi.org/10.1038/s41589-025-01891-7
  27. Front Bioinform. 2025 ;5 1554010
      Single-cell and spatial technologies have transformed our understanding of brain immunology, providing unprecedented insights into immune cell heterogeneity and spatial organisation within the central nervous system. These methods have uncovered complex cellular interactions, rare cell populations, and the dynamic immune landscape in neurological disorders. This review highlights recent advances in single-cell "omics" data analysis and discusses their applicability for brain immunology. Traditional statistical techniques, adapted for single-cell omics, have been crucial in categorizing cell types and identifying gene signatures, overcoming challenges posed by increasingly complex datasets. We explore how machine learning, particularly deep learning methods like autoencoders and graph neural networks, is addressing these challenges by enhancing dimensionality reduction, data integration, and feature extraction. Newly developed foundation models present exciting opportunities for uncovering gene expression programs and predicting genetic perturbations. Focusing on brain development, we demonstrate how single-cell analyses have resolved immune cell heterogeneity, identified temporal maturation trajectories, and uncovered potential therapeutic links to various pathologies, including brain malignancies and neurodegeneration. The integration of single-cell and spatial omics has elucidated the intricate cellular interplay within the developing brain. This mini-review is intended for wet lab biologists at all career stages, offering a concise overview of the evolving landscape of single-cell omics in the age of widely available artificial intelligence.
    Keywords:  data integration; deep learning; development; gene co-expression network; multi-omcis; perturbation prediction; single-cell genomics; trajectory analysis
    DOI:  https://doi.org/10.3389/fbinf.2025.1554010
  28. AAPS PharmSciTech. 2025 Apr 30. 26(5): 121
      Alpelisib, an oral α-specific phosphoinositide 3-kinase (PI3K) inhibitor, has been shown to be safe and effective for some patients with gain-of-function mutation in the PIK3CA oncogene. Alpelisib has received US FDA accelerated approval as Vijoice® film-coated tablets to treat severe PIK3CA-Related Overgrowth Spectrum (PROS). PROS typically displays clinical manifestations in the first year of patient life. Therefore, oral granules were developed as an age-appropriate pediatric dosage form. Bioequivalence between alpelisib granules and tablet and the effect of food on granules pharmacokinetics were assessed in a single-center, randomized, three-treatment, six-sequence, three-period, crossover study among 60 healthy adults. Participants were randomly assigned to receive a single 50-mg alpelisib dose as: (i) tablet following a meal, (ii) granules following a meal, and (iii) granules while fasting. Statistical analysis of non-compartmental pharmacokinetic parameters demonstrated bioequivalence between the 50-mg alpelisib granules and tablet forms when administered with food: estimated geometric mean ratios (90% confidence interval) for granules-versus-tablet area under the curve (AUC) from time zero to infinity (AUCinf), to the last measurable concentration (AUClast) and maximum observed concentration (Cmax) were 0.984 (0.952, 1.02), 0.980 (0.946, 1.02), and 0.947 (0.891, 1.01), respectively. No clinically relevant food effect on 50-mg alpelisib granules pharmacokinetics was observed. These results were accurately predicted using physiologically based biopharmaceutical modeling. Alpelisib granules provide a bioequivalent alternative to tablets for patients prescribed a 50-mg dose and have difficulty swallowing tablets, an important consideration for convenience and compliance of this standard-of-care chronic therapy for patients with PROS. This study was registered in ClinicalTrials.gov on January 4, 2022 (NCT05195892).
    Keywords:  Alpelisib; Bioequivalence; Granules; Pediatric; Physiologically-based biopharmaceutical modeling
    DOI:  https://doi.org/10.1208/s12249-025-03109-4
  29. R Soc Open Sci. 2025 Apr;12(4): 241025
      Different isogenic cells exhibit different responses to the same extracellular signals. Several authors assumed that this variation arose from stochastic signalling noise with the implication that single eukaryotic cells could not detect their surroundings accurately, but work by us and others has shown that the variation is dominated instead by persistent cell-to-cell differences. Here, we analysed previously published data to quantify the sources of variation in pheromone-induced gene expression in Saccharomyces cerevisiae. We found that 91% of response variation was due to stable cell-to-cell differences, 8% from experimental measurement error, and 1% from signalling noise and expression noise. Low noise enabled precise signalling; individual cells could transmit over 3 bits of information through the pheromone response system and so respond differently to eight different pheromone concentrations. Additionally, if individual cells could reference their responses against constitutively expressed proteins, then cells could determine absolute pheromone concentrations with 2 bits of accuracy. These results help explain how individual yeast cells can accurately sense and respond to different extracellular pheromone concentrations.
    Keywords:  cell biology modelling; cell signalling; information theory; systems biology
    DOI:  https://doi.org/10.1098/rsos.241025
  30. Nat Commun. 2025 Apr 28. 16(1): 3906
      The metabolic, immune, and endocrine systems show profound seasonal changes in animals, including humans. In addition, morbidity from cardiovascular and psychiatric diseases is more severe and mortality rate is higher in winter. However, their molecular mechanisms remain unknown. Here we report the seasonal transcriptome of 80 tissues collected over 1 year from male and female rhesus macaques kept in a semi-natural outdoor environment. We find seasonal changes in plasma metabolites and hormones. Transcriptome analysis identifies sex differences in seasonally oscillating genes (SOGs) in all tissues studied, and we generate the web database 'Non-Human Primate Seasonal Transcriptome Atlas (NHPSTA).' Transcriptional regulatory network analysis, siRNA knockdown, and mutant mouse analyses reveal regulation of SOGs by GA-binding protein (GABP). We also demonstrate seasonal oscillations in the expression of disease risk factor genes and drug interacting genes. NHPSTA provides a molecular resource for seasonally regulated physiology and targets for therapeutic interventions for seasonally regulated diseases.
    DOI:  https://doi.org/10.1038/s41467-025-57994-1
  31. Nature. 2025 Apr 30.
      Oncogenic mutations are widespread in normal human tissues1. Similarly, in murine chimeras, cells carrying an oncogenic lesion contribute normal cells to adult tissues without causing cancer2-4. How lineages that escape cancer via normal development differ from the minority that succumb is unclear. Tumours exhibit characteristic cancer hallmarks; we therefore searched for hallmarks that differentiate cancer-prone lineages from resistant lineages. Here we show that total cell cycle duration (Tc) predicts transformation susceptibility across multiple tumour types. Cancer-prone Rb- and p107-deficient retina (Rb is also known as Rb1 and p107 is also known as Rbl1) exhibited defects in apoptosis, senescence, immune surveillance, angiogenesis, DNA repair, polarity and proliferation. Perturbing the SKP2-p27-CDK2/CDK1 axis could block cancer without affecting these hallmarks. Thus, cancer requires more than the presence of its hallmarks. Notably, every tumour-suppressive mutation that we tested increased Tc, and the Tc of the cell of origin of retinoblastoma cells was half that of resistant lineages. Tc also differentiated the cell of origin in Rb-/- pituitary cancer. In lung, loss of Rb and p53 (also known as Trp53) transforms neuroendocrine cells, whereas KrasG12D or BrafV600E mutations transform alveolar type 2 cells5-7. The shortest Tc consistently identified the cell of origin, regardless of mutation timing. Thus, relative Tc is a hallmark of initiation that distinguishes cancer-prone from cancer-resistant lineages in several settings, explaining how mutated cells escape transformation without inducing apoptosis, senescence or immune surveillance.
    DOI:  https://doi.org/10.1038/s41586-025-08935-x
  32. Nature. 2025 May 01.
      
    Keywords:  Biochemistry; CRISPR-Cas9 genome editing; Cell biology
    DOI:  https://doi.org/10.1038/d41586-025-01358-8
  33. EMBO J. 2025 Apr 25.
      Cells change their metabolic profiles in response to underlying gene regulatory networks, but how can alterations in metabolism encode specific transcriptional instructions? Here, we show that forcing a metabolic change in embryonic stem cells (ESCs) promotes a developmental identity that better approximates the inner cell mass (ICM) of the early mammalian blastocyst in cultures. This shift in cellular identity depends on the inhibition of glycolysis and stimulation of oxidative phosphorylation (OXPHOS) triggered by the replacement of D-glucose by D-galactose in ESC media. Enhanced OXPHOS in turn activates NAD + -dependent deacetylases of the Sirtuin family, resulting in the deacetylation of histones and key transcription factors to focus enhancer activity while reducing transcriptional noise, which results in a robustly enhanced ESC phenotype. This exploitation of a NAD + /NADH coenzyme coupled to OXPHOS as a means of programming lineage-specific transcription suggests new paradigms for how cells respond to alterations in their environment, and implies cellular rejuvenation exploits enzymatic activities for simultaneous activation of a discrete enhancer set alongside silencing genome-wide transcriptional noise.
    Keywords:  Aging; Enhancers; Metabolism; Pluripotency; Sirtuins
    DOI:  https://doi.org/10.1038/s44318-025-00417-0
  34. Front Oncol. 2025 ;15 1563985
       Introduction: The insulin receptor (IR) is alternatively spliced into two isoforms, IR-A and IR-B. IR-B is primarily associated with metabolic signaling, whereas IR-A is highly expressed during embryogenesis. IR-A specifically has been associated with several aggressive cancers; however, selective targeting of IR-A has proven difficult due to its homology with IR-B.
    Methods: We generated several antisense oligonucleotides (ASOs) that target the exon 10-12 splice junction site present in IR-A, but not IR-B, mRNA. To test the efficacy of the ASOs, we performed lipofectamine transfections of MDA-MB-231 breast cancer, 22Rv1 prostate carcinoma, and Hs822.T Ewing sarcoma cell lines. We also incubated the MDA-MB-231 cell line with the ASOs in the absence of lipofectamine to determine if they are taken into cells unassisted.
    Results: One ASO variant selectively reduced IR-A mRNA levels with minimal impact on IR-B mRNA and significantly reduced total IR protein. The IR-A ASO successfully induced selective IR-A knockdown in MDA-MB-231 breast cancer cells, which was maintained after a one-week incubation with the ASO. The ASO selectively reduced IR-A mRNA when administered to cells in high doses without the use of a vehicle (i.e. gymnotic delivery). The ASO was also effective at reducing IR-A mRNA in Hs822.T Ewing Sarcoma and 22Rv1 prostate carcinoma cells.
    Discussion: We have developed an ASO that targets IR-A with minimal off-target knockdown of IR-B. We hypothesize that the IR-A ASO will be a useful research tool and may have therapeutic value by inhibiting the oncogenic functions of IR-A in cancer cells.
    Keywords:  IR-A; antisense oligonucleotides; breast cancer; cancer therapeutics; insulin receptor
    DOI:  https://doi.org/10.3389/fonc.2025.1563985
  35. Science. 2025 May;388(6746): eadu9628
      During tissue formation, dynamic cell shape changes drive morphogenesis while asymmetric divisions create cellular diversity. We found that the shifts in cell morphology that shape tissues could concomitantly act as conserved instructive cues that trigger asymmetric division and direct core identity decisions underpinning tissue building. We performed single-cell morphometric analyses of endothelial and other mesenchymal-like cells. Distinct morphological changes switched cells to an "isomorphic" mode of division, which preserved pre-mitotic morphology throughout mitosis. In isomorphic divisions, interphase morphology appeared to provide a geometric code defining mitotic symmetry, fate determinant partitioning, and daughter state. Rab4-positive endosomes recognized this code, allowing them to respond to pre-mitotic morphology and segregate determinants accordingly. Thus, morphogenetic shape change sculpts tissue form while also generating cellular heterogeneity, thereby driving tissue assembly.
    DOI:  https://doi.org/10.1126/science.adu9628
  36. Am J Pathol. 2025 Apr 30. pii: S0002-9440(25)00151-8. [Epub ahead of print]
      Intestinal stem cell (ISC) signaling maintains the balance of self-renewal and differentiation. The role of PI3K signaling in ISC responses to radiation was interrogated using Villin-Cre pik3r1lox/lox (p85ΔIEC) mice and p85α-deficient human enteroids (shp85α). Lethal whole-body irradiation in mice was performed to monitor PI3K-mediated survival responses. Rectal biopsies from patients with radiation proctitis were examined by IHC for the PI3K/Akt- and Wnt-target survivin. The intestinal epithelial cells (IECs) from p85ΔIEC mice showed increased protein levels of p-PTEN, p-AktSer473, survivin, cyclinD1 and ρ-β-cateninSer552 as well as increased mRNA for ISC/PC. In situ hybridization showed that enhanced PI3K signaling reduced Lgr5+ cells but expansion of Axin2+ cells. The shp85α enteroids showed increased mRNA expression of Wnt-targets and transcription factor ASCL2, needed for dedifferentiation-mediated restoration of ablated ISCs. The p85α-deficient enteroids showed reduced HES1 mRNA and increases in secretory (ATOH1/MATH1) signaling determinants GFI1 and SPDEF, indicative of reduced NOTCH signaling. Seahorse analyses and p-p38 staining in IECΔp85 mice indicated that enhanced PI3K signaling led to increased IEC mitochondrial respiration and ROS generation. Expression of survivin correlated with the radiation injury in patients. The current data indicate that PI3K signaling increases mitochondrial ROS generation and ISC activation that improves IEC recovery from radiation-induced injury. The results suggest that increasing PI3K signaling and induced mitochondrial respiration may improve mucosal healing from radiation injury in patients.
    Keywords:  IECs; Mitochondria; PI3K; ROS; Radiation; Stem Cell; p85α
    DOI:  https://doi.org/10.1016/j.ajpath.2025.04.010