bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2024–02–11
fourteen papers selected by
Ralitsa Radostinova Madsen, MRC-PPU
  1. Precision Targeting of Mutant PI3Kα.
  2. Stem cell mTOR signaling directs region-specific cell fate decisions during intestinal nutrient adaptation.
  3. Human vascular organoids with a mosaic AKT1 mutation recapitulate Proteus syndrome.
  4. Epilepsy with faint capillary malformation or reticulated telangiectasia associated with mosaic AKT3 pathogenic variants.
  5. Lineage-specific intolerance to oncogenic drivers restricts histological transformation.
  6. The genetic and dietary landscape of the muscle insulin signalling network.
  7. Genetically Encoded Reporters to Monitor Hypoxia.
  8. Senescent cells and macrophages cooperate through a multi-kinase signaling network to promote intestinal transformation in Drosophila.
  9. CURTAIN-A unique web-based tool for exploration and sharing of MS-based proteomics data.
  10. Self-organizing glycolytic waves fuel cell migration and cancer progression.
  11. Automated single-cell proteomics providing sufficient proteome depth to study complex biology beyond cell type classifications.
  12. Retrospective identification of cell-intrinsic factors that mark pluripotency potential in rare somatic cells.
  13. The cycling and aging mouse female reproductive tract at single-cell resolution.
  14. Simple visualization of submicroscopic protein clusters with a phase-separation-based fluorescent reporter.
  1. Cancer Discov. 2024 Feb 08. 14(2): 204-207
    Precision Targeting of Mutant PI3Kα.
    Grace Q Gong, Bart Vanhaesebroeck.
      PIK3CA, which encodes the p110α catalytic subunit of PI 3-kinase alpha (PI3Kα), is one of the most frequently genetically activated kinases in solid tumors. In two back-to-back papers, Varkaris and colleagues report on the development of a novel allosteric PI3Kα-mutant-selective inhibitor and early clinical experience with this compound. See related article by Varkaris et al., p. 227 (6) . See related article by Varkaris et al., p. 240 (5) .
    DOI:  https://doi.org/10.1158/2159-8290.CD-23-1392
  2. Sci Adv. 2024 Feb 09. 10(6): eadi2671
    Stem cell mTOR signaling directs region-specific cell fate decisions during intestinal nutrient adaptation.
    Jaakko Mattila, Arto Viitanen, Gaia Fabris, Tetiana Strutynska, Jerome Korzelius, Ville Hietakangas.
      The adult intestine is a regionalized organ, whose size and cellular composition are adjusted in response to nutrient status. This involves dynamic regulation of intestinal stem cell (ISC) proliferation and differentiation. How nutrient signaling controls cell fate decisions to drive regional changes in cell-type composition remains unclear. Here, we show that intestinal nutrient adaptation involves region-specific control of cell size, cell number, and differentiation. We uncovered that activation of mTOR complex 1 (mTORC1) increases ISC size in a region-specific manner. mTORC1 activity promotes Delta expression to direct cell fate toward the absorptive enteroblast lineage while inhibiting secretory enteroendocrine cell differentiation. In aged flies, the ISC mTORC1 signaling is deregulated, being constitutively high and unresponsive to diet, which can be mitigated through lifelong intermittent fasting. In conclusion, mTORC1 signaling contributes to the ISC fate decision, enabling regional control of intestinal cell differentiation in response to nutrition.
    DOI:  https://doi.org/10.1126/sciadv.adi2671
  3. bioRxiv. 2024 Jan 27. pii: 2024.01.26.577324. [Epub ahead of print]
    Human vascular organoids with a mosaic AKT1 mutation recapitulate Proteus syndrome.
    Siyu He, Yuefei Zhu, Shradha Chauhan, Daniel Naveed Tavakol, Jong Ha Lee, Rayna Batya-Leia Berris, Cong Xu, Jounghyun H Lee, Caleb Lee, Sarah Cai, Shannon McElroy, Gordana Vunjak-Novakovic, Raju Tomer, Elham Azizi, Bin Xu, Yeh-Hsing Lao, Kam W Leong.
      Vascular malformation, a key clinical phenotype of Proteus syndrome, lacks effective models for pathophysiological study and drug development due to limited patient sample access. To bridge this gap, we built a human vascular organoid model replicating Proteus syndrome's vasculature. Using CRISPR/Cas9 genome editing and gene overexpression, we created induced pluripotent stem cells (iPSCs) embodying the Proteus syndrome-specific AKT E17K point mutation for organoid generation. Our findings revealed that AKT overactivation in these organoids resulted in smaller sizes yet increased vascular connectivity, although with less stable connections. This could be due to the significant vasculogenesis induced by AKT overactivation. This phenomenon likely stems from boosted vasculogenesis triggered by AKT overactivation, leading to increased vascular sprouting. Additionally, a notable increase in dysfunctional PDGFRβ + mural cells, impaired in matrix secretion, was observed in these AKT-overactivated organoids. The application of AKT inhibitors (ARQ092, AZD5363, or GDC0068) reversed the vascular malformations; the inhibitors' effectiveness was directly linked to reduced connectivity in the organoids. In summary, our study introduces an innovative in vitro model combining organoid technology and gene editing to explore vascular pathophysiology in Proteus syndrome. This model not only simulates Proteus syndrome vasculature but also holds potential for mimicking vasculatures of other genetically driven diseases. It represents an advance in drug development for rare diseases, historically plagued by slow progress.
    DOI:  https://doi.org/10.1101/2024.01.26.577324
  4. Am J Med Genet A. 2024 Feb 06. e63551
    Epilepsy with faint capillary malformation or reticulated telangiectasia associated with mosaic AKT3 pathogenic variants.
    Martina De Bortoli, Marta Ivars, Nicole Revencu, Marie-Cécile Nassogne, Cinzia Lavarino, Sonia Paco, Martin Lammens, Anne Renders, Dana Dumitriu, Raphaël Helaers, Laurence M Boon, Eulalia Baselga, Miikka Vikkula.
      Capillary malformations (CMs) are the most common type of vascular anomalies, affecting around 0.3% of newborns. They are usually caused by somatic pathogenic variants in GNAQ or GNA11. PIK3CA and PIK3R1, part of the phosphoinositide 3-kinase-protein kinase B-mammalian target of rapamycin pathway, are mutated in fainter CMs such as diffuse CM with overgrowth and megalencephaly CM. In this study, we present two young patients with a CM-like phenotype associated with cerebral anomalies and severe epilepsy. Pathogenic variants in PIK3CA and PIK3R1, as well as GNAQ and GNA11, were absent in affected cutaneous tissue biopsies. Instead, we identified two somatic pathogenic variants in the AKT3 gene. Subsequent analysis of the DNA obtained from surgically resected brain tissue of one of the two patients confirmed the presence of the AKT3 variant. Focal cortical dysplasia was also detected in this patient. Genetic analysis thus facilitated workup to reach a precise diagnosis for these patients, associating the vascular anomaly with the neurological symptoms. This study underscores the importance of searching for additional signs and symptoms to guide the diagnostic workup, especially in cases with atypical vascular malformations. In addition, it strongly emphasizes the significance of genotype-phenotype correlation studies in guiding clinicians' informed decision-making regarding patient care.
    Keywords:  PI3K signaling; diagnosis; epilepsy; focal cortical dysplasia; genetic; somatic; vascular malformation
    DOI:  https://doi.org/10.1002/ajmg.a.63551
  5. Science. 2024 Feb 09. 383(6683): eadj1415
    Lineage-specific intolerance to oncogenic drivers restricts histological transformation.
    Eric E Gardner, Ethan M Earlie, Kate Li, Jerin Thomas, Melissa J Hubisz, Benjamin D Stein, Chen Zhang, Lewis C Cantley, Ashley M Laughney, Harold Varmus.
      Lung adenocarcinoma (LUAD) and small cell lung cancer (SCLC) are thought to originate from different epithelial cell types in the lung. Intriguingly, LUAD can histologically transform into SCLC after treatment with targeted therapies. In this study, we designed models to follow the conversion of LUAD to SCLC and found that the barrier to histological transformation converges on tolerance to Myc, which we implicate as a lineage-specific driver of the pulmonary neuroendocrine cell. Histological transformations are frequently accompanied by activation of the Akt pathway. Manipulating this pathway permitted tolerance to Myc as an oncogenic driver, producing rare, stem-like cells that transcriptionally resemble the pulmonary basal lineage. These findings suggest that histological transformation may require the plasticity inherent to the basal stem cell, enabling tolerance to previously incompatible oncogenic driver programs.
    DOI:  https://doi.org/10.1126/science.adj1415
  6. Elife. 2024 Feb 08. pii: RP89212. [Epub ahead of print]12
    The genetic and dietary landscape of the muscle insulin signalling network.
    Julian van Gerwen, Stewart W C Masson, Harry B Cutler, Alexis Diaz Vegas, Meg Potter, Jacqueline Stöckli, Søren Madsen, Marin E Nelson, Sean J Humphrey, David E James.
      Metabolic disease is caused by a combination of genetic and environmental factors, yet few studies have examined how these factors influence signal transduction, a key mediator of metabolism. Using mass spectrometry-based phosphoproteomics, we quantified 23,126 phosphosites in skeletal muscle of five genetically distinct mouse strains in two dietary environments, with and without acute in vivo insulin stimulation. Almost half of the insulin-regulated phosphoproteome was modified by genetic background on an ordinary diet, and high-fat high-sugar feeding affected insulin signalling in a strain-dependent manner. Our data revealed coregulated subnetworks within the insulin signalling pathway, expanding our understanding of the pathway's organisation. Furthermore, associating diverse signalling responses with insulin-stimulated glucose uptake uncovered regulators of muscle insulin responsiveness, including the regulatory phosphosite S469 on Pfkfb2, a key activator of glycolysis. Finally, we confirmed the role of glycolysis in modulating insulin action in insulin resistance. Our results underscore the significance of genetics in shaping global signalling responses and their adaptability to environmental changes, emphasising the utility of studying biological diversity with phosphoproteomics to discover key regulatory mechanisms of complex traits.
    Keywords:  computational biology; genetics; insulin resistance; mass spectrometry; metabolism; mouse; phosphoproteomics; rat; signalling; systems biology
    DOI:  https://doi.org/10.7554/eLife.89212
  7. Methods Mol Biol. 2024 ;2755 3-29
    Genetically Encoded Reporters to Monitor Hypoxia.
    Nadine Bauer, Friedemann Kiefer.
      Hypoxia resulting from an imbalance of oxygen availability and consumption defines a metabolic cellular state with a profound impact on developmental processes, tissue maintenance, and the development of pathologies. Fluorescence imaging using genetically encoded reporters enables hypoxia and oxygen imaging with cellular resolution. Thereby unrestricted visualization of hypoxic cells and regions essentially relies on the availability of oxygen-independent fluorescent proteins like UnaG, isolated from the Japanese freshwater eel. Here, we describe the application of recently developed members of a UnaG-based hypoxia reporter family to visualize oxygenation patterns by in vitro live-cell imaging and during the ex vivo analysis of intracranial xenografted tumors. Thus, the generation of stably transfected transgenic tumor cell lines, the in vitro calibration of the genetically encoded sensors, the surgical procedures for orthotopic xenografting of tumors in mice, and workflows for the respective sample preparation and microscopy are outlined.
    Keywords:  FLIM; Fluorescence microscopy; Fluorescent proteins; Genetically encoded sensors; Hypoxia imaging; Hypoxia sensors fluorescent reporters; Live-cell imaging; Oxygen sensing
    DOI:  https://doi.org/10.1007/978-1-0716-3633-6_1
  8. Dev Cell. 2024 Jan 31. pii: S1534-5807(24)00029-7. [Epub ahead of print]
    Senescent cells and macrophages cooperate through a multi-kinase signaling network to promote intestinal transformation in Drosophila.
    Ishwaree Datta, Erdem Bangi.
      Cellular senescence is a conserved biological process that plays a crucial and context-dependent role in cancer. The highly heterogeneous and dynamic nature of senescent cells and their small numbers in tissues make in vivo mechanistic studies of senescence challenging. As a result, how multiple senescence-inducing signals are integrated in vivo to drive senescence in only a small number of cells is unclear. Here, we identify cells that exhibit multiple features of senescence in a Drosophila model of intestinal transformation, which emerge in response to concurrent activation of AKT, JNK, and DNA damage signaling within transformed tissue. Eliminating senescent cells, genetically or by treatment with senolytic compounds, reduces overgrowth and improves survival. We find that senescent cells promote tumorigenesis by recruiting Drosophila macrophages to the transformed tissue, which results in non-autonomous activation of JNK signaling. These findings identify senescent cell-macrophage interactions as an important driver of epithelial transformation.
    Keywords:  Drosophila; cell signaling; colon cancer; hemocyte; macrophage; senescence
    DOI:  https://doi.org/10.1016/j.devcel.2024.01.009
  9. Proc Natl Acad Sci U S A. 2024 Feb 13. 121(7): e2312676121
    CURTAIN-A unique web-based tool for exploration and sharing of MS-based proteomics data.
    Toan K Phung, Kerryn Berndsen, Rosamund Shastry, Tran L C H B Phan, Miratul M K Muqit, Dario R Alessi, Raja S Nirujogi.
      To facilitate analysis and sharing of mass spectrometry (MS)-based proteomics data, we created online tools called CURTAIN (https://curtain.proteo.info) and CURTAIN-PTM (https://curtainptm.proteo.info) with an accompanying series of video tutorials (https://www.youtube.com/@CURTAIN-me6hl). These are designed to enable non-MS experts to interactively peruse volcano plots and deconvolute primary experimental data so that replicates can be visualized in bar charts or violin plots and exported in publication-ready format. They also allow assessment of overall experimental quality by correlation matrix and profile plot analysis. After making a selection of protein "hits", the user can analyze known domain structure, AlphaFold predicted structure, reported interactors, relative expression as well as disease links. CURTAIN-PTM permits analysis of all identified PTM sites on protein(s) of interest with selected databases. CURTAIN-PTM also links with the Kinase Library to predict upstream kinases that may phosphorylate sites of interest. We provide examples of the utility of CURTAIN and CURTAIN-PTM in analyzing how targeted degradation of the PPM1H Rab phosphatase that counteracts the Parkinson's LRRK2 kinase impacts cellular protein levels and phosphorylation sites. We also reanalyzed a ubiquitylation dataset, characterizing the PINK1-Parkin pathway activation in primary neurons, revealing data of interest not highlighted previously. CURTAIN and CURTAIN-PTM are free to use and open source, enabling researchers to share and maximize the impact of their proteomics data. We advocate that MS data published in volcano plot format be reported containing a shareable CURTAIN weblink, thereby allowing readers to better analyze and exploit the data.
    Keywords:  PTM; bioinformatics; mass spectrometry; proteomics; sharing data
    DOI:  https://doi.org/10.1073/pnas.2312676121
  10. bioRxiv. 2024 Jan 28. pii: 2024.01.28.577603. [Epub ahead of print]
    Self-organizing glycolytic waves fuel cell migration and cancer progression.
    Huiwang Zhan, Dhiman Sankar Pal, Jane Borleis, Chris Janetopoulos, Chuan-Hsiang Huang, Peter N Devreotes.
      Glycolysis has traditionally been thought to take place in the cytosol but we observed the enrichment of glycolytic enzymes in propagating waves of the cell cortex in human epithelial cells. These waves reflect excitable Ras/PI3K signal transduction and F-actin/actomyosin networks that drive cellular protrusions, suggesting that localized glycolysis at the cortex provides ATP for cell morphological events such as migration, phagocytosis, and cytokinesis. Perturbations that altered cortical waves caused corresponding changes in enzyme localization and ATP production whereas synthetic recruitment of glycolytic enzymes to the cell cortex enhanced cell spreading and motility. Interestingly, the cortical waves and ATP levels were positively correlated with the metastatic potential of cancer cells. The coordinated signal transduction, cytoskeletal, and glycolytic waves in cancer cells may explain their increased motility and their greater reliance on glycolysis, often referred to as the Warburg effect.
    DOI:  https://doi.org/10.1101/2024.01.28.577603
  11. bioRxiv. 2024 Jan 22. pii: 2024.01.20.576369. [Epub ahead of print]
    Automated single-cell proteomics providing sufficient proteome depth to study complex biology beyond cell type classifications.
    Claudia Ctortecka, Natalie M Clark, Brian Boyle, Anjali Seth, D R Mani, Namrata D Udeshi, Steven A Carr.
      Mass spectrometry (MS)-based single-cell proteomics (SCP) has gained massive attention as a viable complement to other single cell approaches. The rapid technological and computational advances in the field have pushed the boundaries of sensitivity and throughput. However, reproducible quantification of thousands of proteins within a single cell at reasonable proteome depth to characterize biological phenomena remains a challenge. To address some of those limitations we present a combination of fully automated single cell sample preparation utilizing a dedicated chip within the picolitre dispensing robot, the cellenONE. The proteoCHIP EVO 96 can be directly interfaced with the Evosep One chromatographic system for in-line desalting and highly reproducible separation with a throughput of 80 samples per day. This, in combination with the Bruker timsTOF MS instruments, demonstrates double the identifications without manual sample handling. Moreover, relative to standard high-performance liquid chromatography, the Evosep One separation provides further 2-fold improvement in protein identifications. The implementation of the newest generation timsTOF Ultra with our proteoCHIP EVO 96-based sample preparation workflow reproducibly identifies up to 4,000 proteins per single HEK-293T without a carrier or match-between runs. Our current SCP depth spans over 4 orders of magnitude and identifies over 50 biologically relevant ubiquitin ligases. We complement our highly reproducible single-cell proteomics workflow to profile hundreds of lipopolysaccharide (LPS)-perturbed THP-1 cells and identified key regulatory proteins involved in interleukin and interferon signaling. This study demonstrates that the proteoCHIP EVO 96-based SCP sample preparation with the timsTOF Ultra provides sufficient proteome depth to study complex biology beyond cell-type classifications.
    DOI:  https://doi.org/10.1101/2024.01.20.576369
  12. Cell Syst. 2024 Jan 30. pii: S2405-4712(24)00022-X. [Epub ahead of print]
    Retrospective identification of cell-intrinsic factors that mark pluripotency potential in rare somatic cells.
    Naveen Jain, Yogesh Goyal, Margaret C Dunagin, Christopher J Cote, Ian A Mellis, Benjamin Emert, Connie L Jiang, Ian P Dardani, Sam Reffsin, Miles Arnett, Wenli Yang, Arjun Raj.
      Pluripotency can be induced in somatic cells by the expression of OCT4, KLF4, SOX2, and MYC. Usually only a rare subset of cells reprogram, and the molecular characteristics of this subset remain unknown. We apply retrospective clone tracing to identify and characterize the rare human fibroblasts primed for reprogramming. These fibroblasts showed markers of increased cell cycle speed and decreased fibroblast activation. Knockdown of a fibroblast activation factor identified by our analysis increased the reprogramming efficiency. We provide evidence for a unified model in which cells can move into and out of the primed state over time, explaining how reprogramming appears deterministic at short timescales and stochastic at long timescales. Furthermore, inhibiting the activity of LSD1 enlarged the pool of cells that were primed for reprogramming. Thus, even homogeneous cell populations can exhibit heritable molecular variability that can dictate whether individual rare cells will reprogram or not.
    Keywords:  cellular barcoding; iPSC reprogramming; lineage tracing; systems biology
    DOI:  https://doi.org/10.1016/j.cels.2024.01.001
  13. Cell. 2024 Jan 31. pii: S0092-8674(24)00058-8. [Epub ahead of print]
    The cycling and aging mouse female reproductive tract at single-cell resolution.
    Ivana Winkler, Alexander Tolkachov, Fritjof Lammers, Perrine Lacour, Klaudija Daugelaite, Nina Schneider, Marie-Luise Koch, Jasper Panten, Florian Grünschläger, Tanja Poth, Bianca Machado de Ávila, Augusto Schneider, Simon Haas, Duncan T Odom, Ângela Gonçalves.
      The female reproductive tract (FRT) undergoes extensive remodeling during reproductive cycling. This recurrent remodeling and how it shapes organ-specific aging remains poorly explored. Using single-cell and spatial transcriptomics, we systematically characterized morphological and gene expression changes occurring in ovary, oviduct, uterus, cervix, and vagina at each phase of the mouse estrous cycle, during decidualization, and into aging. These analyses reveal that fibroblasts play central-and highly organ-specific-roles in FRT remodeling by orchestrating extracellular matrix (ECM) reorganization and inflammation. Our results suggest a model wherein recurrent FRT remodeling over reproductive lifespan drives the gradual, age-related development of fibrosis and chronic inflammation. This hypothesis was directly tested using chemical ablation of cycling, which reduced fibrotic accumulation during aging. Our atlas provides extensive detail into how estrus, pregnancy, and aging shape the organs of the female reproductive tract and reveals the unexpected cost of the recurrent remodeling required for reproduction.
    Keywords:  aging; cervix; fibrosis; inflammation; ovary; oviduct; single cell; spatial; uterus; vagina
    DOI:  https://doi.org/10.1016/j.cell.2024.01.021
  14. Cell Syst. 2024 Feb 05. pii: S2405-4712(24)00026-7. [Epub ahead of print]
    Simple visualization of submicroscopic protein clusters with a phase-separation-based fluorescent reporter.
    Thomas R Mumford, Diarmid Rae, Emily Brackhahn, Abbas Idris, David Gonzalez-Martinez, Ayush Aditya Pal, Michael C Chung, Juan Guan, Elizabeth Rhoades, Lukasz J Bugaj.
      Protein clustering plays numerous roles in cell physiology and disease. However, protein oligomers can be difficult to detect because they are often too small to appear as puncta in conventional fluorescence microscopy. Here, we describe a fluorescent reporter strategy that detects protein clusters with high sensitivity called CluMPS (clusters magnified by phase separation). A CluMPS reporter detects and visually amplifies even small clusters of a binding partner, generating large, quantifiable fluorescence condensates. We use computational modeling and optogenetic clustering to demonstrate that CluMPS can detect small oligomers and behaves rationally according to key system parameters. CluMPS detected small aggregates of pathological proteins where the corresponding GFP fusions appeared diffuse. CluMPS also detected and tracked clusters of unmodified and tagged endogenous proteins, and orthogonal CluMPS probes could be multiplexed in cells. CluMPS provides a powerful yet straightforward approach to observe higher-order protein assembly in its native cellular context. A record of this paper's transparent peer review process is included in the supplemental information.
    Keywords:  clusters; condensates; fluorescent; oligomers; optogenetics; phase-separation; puncta; reporter; sensor; stochastic modeling
    DOI:  https://doi.org/10.1016/j.cels.2024.01.005
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