bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2024–02–04
eleven papers selected by
Ralitsa Radostinova Madsen, MRC-PPU
  1. A robust and inducible precise genome editing via an all-in-one prime editor in human pluripotent stem cells.
  2. A prime editor that makes space for insertions.
  3. mTORC1 controls murine postprandial hepatic glycogen synthesis via Ppp1r3b.
  4. Loss of Pip4k2c confers liver-metastatic organotropism through insulin-dependent PI3K-AKT pathway activation.
  5. The ability to sense the environment is heterogeneously distributed in cell populations.
  6. An optimized toolkit for prime editing.
  7. Single-cell morphodynamical trajectories enable prediction of gene expression accompanying cell state change.
  8. Comprehensive multiscale analysis of lactate metabolic dynamics in vitro and in vivo using highly responsive biosensors.
  9. CRISPR technologies for genome, epigenome and transcriptome editing.
  10. Scbean: a python library for single-cell multi-omics data analysis.
  11. Logic-based modeling of biological networks with Netflux.
  1. bioRxiv. 2024 Jan 19. pii: 2024.01.18.576233. [Epub ahead of print]
    A robust and inducible precise genome editing via an all-in-one prime editor in human pluripotent stem cells.
    Youjun Wu, Aaron Zhong, Mega Sidharta, Tae Wan Kim, Bernny Ramirez, Benjamin Persily, Lorenz Studer, Ting Zhou.
      Prime editing (PE) allows for precise genome editing in human pluripotent stem cells (hPSCs), such as introducing single nucleotide modifications, small deletions, or insertions at a specific genomic locus, a strategy that shows great promise for creating "Disease in a dish" models. To improve the effectiveness of prime editing in hPSCs, we systematically compared and combined the "inhibition of mismatch repair pathway and p53" on top of the "PEmax" to generate an all-in-one "PE-Plus" prime editor. We show that PE-Plus conducts the most efficient editing among the current PE tools in hPSCs. We further established an inducible prime editing platform in hPSCs by incorporating the all-in-one PE vector into a safe-harbor locus and demonstrated temporal control of precise editing in both hPSCs and differentiated cells. By evaluating disease-associated mutations, we show that this platform allows efficient creation of both monoallelic and biallelic disease-relevant mutations in hPSCs. In addition, this platform enables the efficient introduction of single or multiple edits in one step, demonstrating potential for multiplex editing. Therefore, our method presents an efficient and controllable multiplex prime editing tool in hPSCs and their differentiated progeny.
    DOI:  https://doi.org/10.1101/2024.01.18.576233
  2. Nat Methods. 2024 Feb 01.
    A prime editor that makes space for insertions.
      
    DOI:  https://doi.org/10.1038/s41592-023-02163-9
  3. J Clin Invest. 2024 Jan 30. pii: e173782. [Epub ahead of print]
    mTORC1 controls murine postprandial hepatic glycogen synthesis via Ppp1r3b.
    Kahealani Uehara, Won Dong Lee, Megan Stefkovich, Dipsikha Biswas, Dominic Santoleri, Anna E Garcia Whitlock, William J Quinn Iii, Talia N Coopersmith, Kate Townsend Creasy, Daniel J Rader, Kei Sakamoto, Joshua D Rabinowitz, Paul M Titchenell.
      In response to a meal, insulin drives hepatic glycogen synthesis to help regulate systemic glucose homeostasis. The mechanistic target of rapamycin complex 1 (mTORC1) is a well-established insulin target and contributes to the postprandial control of liver lipid metabolism, autophagy, and protein synthesis. However, its role in hepatic glucose metabolism is less understood. Here, we used metabolomics, isotope tracing, and mouse genetics to define a role for liver mTORC1 signaling in the control of postprandial glycolytic intermediates and glycogen deposition. We show that mTORC1 is required for glycogen synthase activity and glycogenesis. Mechanistically, hepatic mTORC1 activity promotes the feeding-dependent induction of Ppp1r3b, a gene encoding a phosphatase important for glycogen synthase activity whose polymorphisms are linked to human diabetes. Re-expression of Ppp1r3b in livers lacking mTORC1 signaling enhances glycogen synthase activity and restores postprandial glycogen content. mTORC1-dependent transcriptional control of Ppp1r3b is facilitated by FOXO1, a well characterized transcriptional regulator involved in the hepatic response to nutrient intake. Collectively, we identify a role for mTORC1 signaling in the transcriptional regulation of Ppp1r3b and the subsequent induction of postprandial hepatic glycogen synthesis.
    Keywords:  Endocrinology; Glucose metabolism; Insulin signaling; Metabolism
    DOI:  https://doi.org/10.1172/JCI173782
  4. Nat Cancer. 2024 Jan 29.
    Loss of Pip4k2c confers liver-metastatic organotropism through insulin-dependent PI3K-AKT pathway activation.
    Meri Rogava, Tyler J Aprati, Wei-Yu Chi, Johannes C Melms, Clemens Hug, Stephanie H Davis, Ethan M Earlie, Charlie Chung, Sachin K Deshmukh, Sharon Wu, George Sledge, Stephen Tang, Patricia Ho, Amit Dipak Amin, Lindsay Caprio, Carino Gurjao, Somnath Tagore, Bryan Ngo, Michael J Lee, Giorgia Zanetti, Yiping Wang, Sean Chen, William Ge, Luiza Martins Nascentes Melo, Gabriele Allies, Jonas Rösler, Goeffrey T Gibney, Oliver J Schmitz, Megan Sykes, Rémi J Creusot, Thomas Tüting, Dirk Schadendorf, Martin Röcken, Thomas K Eigentler, Andrei Molotkov, Akiva Mintz, Samuel F Bakhoum, Semir Beyaz, Lewis C Cantley, Peter K Sorger, Sven W Meckelmann, Alpaslan Tasdogan, David Liu, Ashley M Laughney, Benjamin Izar.
      Liver metastasis (LM) confers poor survival and therapy resistance across cancer types, but the mechanisms of liver-metastatic organotropism remain unknown. Here, through in vivo CRISPR-Cas9 screens, we found that Pip4k2c loss conferred LM but had no impact on lung metastasis or primary tumor growth. Pip4k2c-deficient cells were hypersensitized to insulin-mediated PI3K/AKT signaling and exploited the insulin-rich liver milieu for organ-specific metastasis. We observed concordant changes in PIP4K2C expression and distinct metabolic changes in 3,511 patient melanomas, including primary tumors, LMs and lung metastases. We found that systemic PI3K inhibition exacerbated LM burden in mice injected with Pip4k2c-deficient cancer cells through host-mediated increase in hepatic insulin levels; however, this circuit could be broken by concurrent administration of an SGLT2 inhibitor or feeding of a ketogenic diet. Thus, this work demonstrates a rare example of metastatic organotropism through co-optation of physiological metabolic cues and proposes therapeutic avenues to counteract these mechanisms.
    DOI:  https://doi.org/10.1038/s43018-023-00704-x
  5. Elife. 2024 Jan 31. pii: RP87747. [Epub ahead of print]12
    The ability to sense the environment is heterogeneously distributed in cell populations.
    Andrew Goetz, Hoda Akl, Purushottam Dixit.
      Channel capacity of signaling networks quantifies their fidelity in sensing extracellular inputs. Low estimates of channel capacities for several mammalian signaling networks suggest that cells can barely detect the presence/absence of environmental signals. However, given the extensive heterogeneity and temporal stability of cell state variables, we hypothesize that the sensing ability itself may depend on the state of the cells. In this work, we present an information-theoretic framework to quantify the distribution of sensing abilities from single-cell data. Using data on two mammalian pathways, we show that sensing abilities are widely distributed in the population and most cells achieve better resolution of inputs compared to an 'average cell'. We verify these predictions using live-cell imaging data on the IGFR/FoxO pathway. Importantly, we identify cell state variables that correlate with cells' sensing abilities. This information-theoretic framework will significantly improve our understanding of how cells sense in their environment.
    Keywords:  human; information transfer; maximum entropy; physics of living systems; signaling networks
    DOI:  https://doi.org/10.7554/eLife.87747
  6. Nat Biotechnol. 2024 Jan 29.
    An optimized toolkit for prime editing.
    Ryan M Marks, Ori Scott, Evgueni A Ivakine, Ronald D Cohn.
      
    DOI:  https://doi.org/10.1038/s41587-023-02091-1
  7. bioRxiv. 2024 Jan 19. pii: 2024.01.18.576248. [Epub ahead of print]
    Single-cell morphodynamical trajectories enable prediction of gene expression accompanying cell state change.
    Jeremy Copperman, Ian C Mclean, Sean M Gross, Young Hwan Chang, Daniel M Zuckerman, Laura M Heiser.
      Extracellular signals induce changes to molecular programs that modulate multiple cellular phenotypes, including proliferation, motility, and differentiation status. The connection between dynamically adapting phenotypic states and the molecular programs that define them is not well understood. Here we develop data-driven models of single-cell phenotypic responses to extracellular stimuli by linking gene transcription levels to "morphodynamics" - changes in cell morphology and motility observable in time-lapse image data. We adopt a dynamics-first view of cell state by grouping single-cell trajectories into states with shared morphodynamic responses. The single-cell trajectories enable development of a first-of-its-kind computational approach to map live-cell dynamics to snapshot gene transcript levels, which we term MMIST, Molecular and Morphodynamics-Integrated Single-cell Trajectories. The key conceptual advance of MMIST is that cell behavior can be quantified based on dynamically defined states and that extracellular signals alter the overall distribution of cell states by altering rates of switching between states. We find a cell state landscape that is bound by epithelial and mesenchymal endpoints, with distinct sequences of epithelial to mesenchymal transition (EMT) and mesenchymal to epithelial transition (MET) intermediates. The analysis yields predictions for gene expression changes consistent with curated EMT gene sets and provides a prediction of thousands of RNA transcripts through extracellular signal-induced EMT and MET with near-continuous time resolution. The MMIST framework leverages true single-cell dynamical behavior to generate molecular-level omics inferences and is broadly applicable to other biological domains, time-lapse imaging approaches and molecular snapshot data.
    DOI:  https://doi.org/10.1101/2024.01.18.576248
  8. Nat Protoc. 2024 Feb 02.
    Comprehensive multiscale analysis of lactate metabolic dynamics in vitro and in vivo using highly responsive biosensors.
    Aoxue Wang, Yejun Zou, Shuning Liu, Xiuze Zhang, Ting Li, Lijuan Zhang, Ruwen Wang, Yale Xia, Xie Li, Zhuo Zhang, Tiemin Liu, Zhenyu Ju, Ru Wang, Joseph Loscalzo, Yi Yang, Yuzheng Zhao.
      As a key glycolytic metabolite, lactate has a central role in diverse physiological and pathological processes. However, comprehensive multiscale analysis of lactate metabolic dynamics in vitro and in vivo has remained an unsolved problem until now owing to the lack of a high-performance tool. We recently developed a series of genetically encoded fluorescent sensors for lactate, named FiLa, which illuminate lactate metabolism in cells, subcellular organelles, animals, and human serum and urine. In this protocol, we first describe the FiLa sensor-based strategies for real-time subcellular bioenergetic flux analysis by profiling the lactate metabolic response to different nutritional and pharmacological conditions, which provides a systematic-level view of cellular metabolic function at the subcellular scale for the first time. We also report detailed procedures for imaging lactate dynamics in live mice through a cell microcapsule system or recombinant adeno-associated virus and for the rapid and simple assay of lactate in human body fluids. This comprehensive multiscale metabolic analysis strategy may also be applied to other metabolite biosensors using various analytic platforms, further expanding its usability. The protocol is suited for users with expertise in biochemistry, molecular biology and cell biology. Typically, the preparation of FiLa-expressing cells or mice takes 2 days to 4 weeks, and live-cell and in vivo imaging can be performed within 1-2 hours. For the FiLa-based assay of body fluids, the whole measuring procedure generally takes ~1 min for one sample in a manual assay or ~3 min for 96 samples in an automatic microplate assay.
    DOI:  https://doi.org/10.1038/s41596-023-00948-y
  9. Nat Rev Mol Cell Biol. 2024 Feb 02.
    CRISPR technologies for genome, epigenome and transcriptome editing.
    Lukas Villiger, Julia Joung, Luke Koblan, Jonathan Weissman, Omar O Abudayyeh, Jonathan S Gootenberg.
      Our ability to edit genomes lags behind our capacity to sequence them, but the growing understanding of CRISPR biology and its application to genome, epigenome and transcriptome engineering is narrowing this gap. In this Review, we discuss recent developments of various CRISPR-based systems that can transiently or permanently modify the genome and the transcriptome. The discovery of further CRISPR enzymes and systems through functional metagenomics has meaningfully broadened the applicability of CRISPR-based editing. Engineered Cas variants offer diverse capabilities such as base editing, prime editing, gene insertion and gene regulation, thereby providing a panoply of tools for the scientific community. We highlight the strengths and weaknesses of current CRISPR tools, considering their efficiency, precision, specificity, reliance on cellular DNA repair mechanisms and their applications in both fundamental biology and therapeutics. Finally, we discuss ongoing clinical trials that illustrate the potential impact of CRISPR systems on human health.
    DOI:  https://doi.org/10.1038/s41580-023-00697-6
  10. Bioinformatics. 2024 Jan 30. pii: btae053. [Epub ahead of print]
    Scbean: a python library for single-cell multi-omics data analysis.
    Haohui Zhang, Yuwei Wang, Bin Lian, Yiran Wang, Xingyi Li, Tao Wang, Xuequn Shang, Hui Yang, Ahmad Aziz, Jialu Hu.
      Single-cell multi-omics technologies provide a unique platform for characterizing cell states and reconstructing developmental process by simultaneously quantifying and integrating molecular signatures across various modalities, including genome, transcriptome, epigenome and other omics layers. However, there is still an urgent unmet need for novel computational tools in this nascent field, which are critical for both effective and efficient interrogation of functionality across different omics modalities. Scbean represents a user-friendly Python library, designed to seamlessly incorporate a diverse array of models for the examination of single-cell data, encompassing both paired and unpaired multi-omics data. The library offers uniform and straightforward interfaces for tasks such as dimensionality reduction, batch effect elimination, cell label transfer from well-annotated scRNA-seq data to scATAC-seq data, and the identification of spatially variable genes. Moreover, Scbean's models are engineered to harness the computational power of GPU acceleration through Tensorflow, rendering them capable of effortlessly handling datasets comprising millions of cells.
    AVAILABILITY: Scbean is released on the Python Package Index (PyPI) (https://pypi.org/project/scbean/) and GitHub (https://github.com/jhu99/scbean) under the MIT license. The documentation and example code can be found at https://scbean.readthedocs.io/en/latest/.
    DOI:  https://doi.org/10.1093/bioinformatics/btae053
  11. bioRxiv. 2024 Jan 15. pii: 2024.01.11.575227. [Epub ahead of print]
    Logic-based modeling of biological networks with Netflux.
    Alexander P Clark, Mukti Chowkwale, Alexander Paap, Stephen Dang, Jeffrey J Saucerman.
      Molecular signaling networks drive a diverse range of cellular decisions, including whether to proliferate, how and when to die, and many processes in between. Such networks often connect hundreds of proteins, genes, and processes. Understanding these complex networks is greatly aided by computational modeling, but these tools require extensive programming knowledge. In this article, we describe a user-friendly, programming-free network simulation tool called Netflux ( https://github.com/saucermanlab/Netflux ). Over the last decade, Netflux has been used to construct numerous predictive network models that have deepened our understanding of how complex biological networks make cell decisions. Here, we provide a Netflux tutorial that covers how to construct a network model and then simulate network responses to perturbations. Upon completion of this tutorial, you will be able to construct your own model in Netflux and simulate how perturbations to proteins and genes propagate through signaling and gene-regulatory networks.
    DOI:  https://doi.org/10.1101/2024.01.11.575227
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