bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2023–05–28
23 papers selected by
Ralitsa Radostinova Madsen, MRC-PPU
  1. Hyperphosphorylated PTEN exerts oncogenic properties.
  2. A small-molecule PI3Kα activator for cardioprotection and neuroregeneration.
  3. pYtags enable spatiotemporal measurements of receptor tyrosine kinase signaling in living cells.
  4. The E2 ubiquitin-conjugating enzymes UBE2D1 and UBE2D2 regulate VEGFR2 dynamics and endothelial function.
  5. Loss of PI3k activity of inositol polyphosphate multikinase impairs PDK1-mediated AKT activation, cell migration, and intestinal homeostasis.
  6. Actuation of single downstream nodes in growth factor network steers immune cell migration.
  7. AKT activation because of PTEN loss upregulates xCT via GSK3β/NRF2, leading to inhibition of ferroptosis in PTEN-mutant tumor cells.
  8. Dual inhibition of phosphoinositide 3-kinases delta and gamma reduces chronic B cell activation and autoantibody production in a mouse model of lupus.
  9. Mechanosensitive stem cell fate choice is instructed by dynamic fluctuations in activation of Rho GTPases.
  10. Feature selection for preserving biological trajectories in single-cell data.
  11. High-content CRISPR screening.
  12. Genetically encoded photocatalytic protein labeling enables spatially-resolved profiling of intracellular proteome.
  13. LFQ-Based Peptide and Protein Intensity Differential Expression Analysis.
  14. Stabilized mosaic single-cell data integration using unshared features.
  15. Strategies for precise gene edits in mammalian cells.
  16. Efficient precise integration of large DNA sequences with 3'-overhang dsDNA donors using CRISPR/Cas9.
  17. Filter inference: A scalable nonlinear mixed effects inference approach for snapshot time series data.
  18. Dictionary learning for integrative, multimodal and scalable single-cell analysis.
  19. Unbiased evaluation of rapamycin's specificity as an mTOR inhibitor.
  20. Discovery and Validation of Context-Dependent Synthetic Mammalian Promoters.
  21. The mitochondrial genome as a modifier of autism versus cancer phenotypes in PTEN hamartoma tumor syndrome.
  22. Spatial single cell metabolomics: Current challenges and future developments.
  23. Nearly maximal information gain due to time integration in central dogma reactions.
  1. Nat Commun. 2023 May 24. 14(1): 2983
    Hyperphosphorylated PTEN exerts oncogenic properties.
    Janine H van Ree, Karthik B Jeganathan, Raul O Fierro Velasco, Cheng Zhang, Ismail Can, Masakazu Hamada, Hu Li, Darren J Baker, Jan M van Deursen.
      PTEN is a multifaceted tumor suppressor that is highly sensitive to alterations in expression or function. The PTEN C-tail domain, which is rich in phosphorylation sites, has been implicated in PTEN stability, localization, catalytic activity, and protein interactions, but its role in tumorigenesis remains unclear. To address this, we utilized several mouse strains with nonlethal C-tail mutations. Mice homozygous for a deletion that includes S370, S380, T382 and T383 contain low PTEN levels and hyperactive AKT but are not tumor prone. Analysis of mice containing nonphosphorylatable or phosphomimetic versions of S380, a residue hyperphosphorylated in human gastric cancers, reveal that PTEN stability and ability to inhibit PI3K-AKT depends on dynamic phosphorylation-dephosphorylation of this residue. While phosphomimetic S380 drives neoplastic growth in prostate by promoting nuclear accumulation of β-catenin, nonphosphorylatable S380 is not tumorigenic. These data suggest that C-tail hyperphosphorylation creates oncogenic PTEN and is a potential target for anti-cancer therapy.
    DOI:  https://doi.org/10.1038/s41467-023-38740-x
  2. Nature. 2023 May 24.
    A small-molecule PI3Kα activator for cardioprotection and neuroregeneration.
    Grace Q Gong, Benoit Bilanges, Ben Allsop, Glenn R Masson, Victoria Roberton, Trevor Askwith, Sally Oxenford, Ralitsa R Madsen, Sarah E Conduit, Dom Bellini, Martina Fitzek, Matt Collier, Osman Najam, Zhenhe He, Ben Wahab, Stephen H McLaughlin, A W Edith Chan, Isabella Feierberg, Andrew Madin, Daniele Morelli, Amandeep Bhamra, Vanesa Vinciauskaite, Karen E Anderson, Silvia Surinova, Nikos Pinotsis, Elena Lopez-Guadamillas, Matthew Wilcox, Alice Hooper, Chandni Patel, Maria A Whitehead, Tom D Bunney, Len R Stephens, Phillip T Hawkins, Matilda Katan, Derek M Yellon, Sean M Davidson, David M Smith, James B Phillips, Richard Angell, Roger L Williams, Bart Vanhaesebroeck.
      Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development1-5. This also applies to the PI3K signalling pathway, which has been extensively targeted by inhibitors for conditions with PI3K overactivation, such as cancer and immune dysregulation. Here we report the discovery of UCL-TRO-1938 (referred to as 1938 hereon), a small-molecule activator of the PI3Kα isoform, a crucial effector of growth factor signalling. 1938 allosterically activates PI3Kα through a distinct mechanism by enhancing multiple steps of the PI3Kα catalytic cycle and causes both local and global conformational changes in the PI3Kα structure. This compound is selective for PI3Kα over other PI3K isoforms and multiple protein and lipid kinases. It transiently activates PI3K signalling in all rodent and human cells tested, resulting in cellular responses such as proliferation and neurite outgrowth. In rodent models, acute treatment with 1938 provides cardioprotection from ischaemia-reperfusion injury and, after local administration, enhances nerve regeneration following nerve crush. This study identifies a chemical tool to directly probe the PI3Kα signalling pathway and a new approach to modulate PI3K activity, widening the therapeutic potential of targeting these enzymes through short-term activation for tissue protection and regeneration. Our findings illustrate the potential of activating kinases for therapeutic benefit, a currently largely untapped area of drug development.
    DOI:  https://doi.org/10.1038/s41586-023-05972-2
  3. Elife. 2023 05 22. pii: e82863. [Epub ahead of print]12
    pYtags enable spatiotemporal measurements of receptor tyrosine kinase signaling in living cells.
    Payam E Farahani, Xiaoyu Yang, Emily V Mesev, Kaylan A Fomby, Ellen H Brumbaugh-Reed, Caleb J Bashor, Celeste M Nelson, Jared E Toettcher.
      Receptor tyrosine kinases (RTKs) are major signaling hubs in metazoans, playing crucial roles in cell proliferation, migration, and differentiation. However, few tools are available to measure the activity of a specific RTK in individual living cells. Here, we present pYtags, a modular approach for monitoring the activity of a user-defined RTK by live-cell microscopy. pYtags consist of an RTK modified with a tyrosine activation motif that, when phosphorylated, recruits a fluorescently labeled tandem SH2 domain with high specificity. We show that pYtags enable the monitoring of a specific RTK on seconds-to-minutes time scales and across subcellular and multicellular length scales. Using a pYtag biosensor for epidermal growth factor receptor (EGFR), we quantitatively characterize how signaling dynamics vary with the identity and dose of activating ligand. We show that orthogonal pYtags can be used to monitor the dynamics of EGFR and ErbB2 activity in the same cell, revealing distinct phases of activation for each RTK. The specificity and modularity of pYtags open the door to robust biosensors of multiple tyrosine kinases and may enable engineering of synthetic receptors with orthogonal response programs.
    Keywords:  biosensor; cell biology; cell signaling; human; map kinase; mouse; receptor tyrosine kinase; signaling dynamics; tyrosine phosphorylation
    DOI:  https://doi.org/10.7554/eLife.82863
  4. J Cell Sci. 2023 May 15. pii: jcs260657. [Epub ahead of print]136(10):
    The E2 ubiquitin-conjugating enzymes UBE2D1 and UBE2D2 regulate VEGFR2 dynamics and endothelial function.
    William R Critchley, Gina A Smith, Ian C Zachary, Michael A Harrison, Sreenivasan Ponnambalam.
      Vascular endothelial growth factor receptor 2 (VEGFR2, encoded by KDR) regulates endothelial function and angiogenesis. VEGFR2 undergoes ubiquitination that programs this receptor for trafficking and proteolysis, but the ubiquitin-modifying enzymes involved are ill-defined. Herein, we used a reverse genetics screen for the human E2 family of ubiquitin-conjugating enzymes to identify gene products that regulate VEGFR2 ubiquitination and proteolysis. We found that depletion of either UBE2D1 or UBE2D2 in endothelial cells caused a rise in steady-state VEGFR2 levels. This rise in plasma membrane VEGFR2 levels impacted on VEGF-A-stimulated signalling, with increased activation of canonical MAPK, phospholipase Cγ1 and Akt pathways. Analysis of biosynthetic VEGFR2 is consistent with a role for UBE2D enzymes in influencing plasma membrane VEGFR2 levels. Cell-surface-specific biotinylation and recycling studies showed an increase in VEGFR2 recycling to the plasma membrane upon reduction in UBE2D levels. Depletion of either UBE2D1 or UBE2D2 stimulated endothelial tubulogenesis, which is consistent with increased VEGFR2 plasma membrane levels promoting the cellular response to exogenous VEGF-A. Our studies identify a key role for UBE2D1 and UBE2D2 in regulating VEGFR2 function in angiogenesis.
    Keywords:  Angiogenesis; Endothelial cells; Signalling; UBE2D1; UBE2D2; Ubiquitin; VEGFR2
    DOI:  https://doi.org/10.1242/jcs.260657
  5. iScience. 2023 May 19. 26(5): 106623
    Loss of PI3k activity of inositol polyphosphate multikinase impairs PDK1-mediated AKT activation, cell migration, and intestinal homeostasis.
    Luke Reilly, Evan R Semenza, George Koshkaryan, Subrata Mishra, Sujan Chatterjee, Efrat Abramson, Pamela Mishra, Yoshitasu Sei, Stephen A Wank, Mark Donowitz, Solomon H Snyder, Prasun Guha.
      Protein kinase B (AKT) is essential for cell survival, proliferation, and migration and has been associated with several diseases. Here, we demonstrate that inositol polyphosphate multikinase (IPMK's) lipid kinase property drives AKT activation via increasing membrane localization and activation of PDK1 (3-Phosphoinositide-dependent kinase 1), largely independent of class I PI3k (cPI3K). Deletion of IPMK impairs cell migration, which is partially associated with the abolition of PDK1-mediated ROCK1 disinhibition and subsequent myosin light chain (MLC) phosphorylation. IPMK is highly expressed in intestinal epithelial cells (IEC). Deleting IPMK in IEC reduced AKT phosphorylation and diminished the number of Paneth cells. Ablation of IPMK impaired IEC regeneration both basally and after chemotherapy-induced damage, suggesting a broad role for IPMK in activating AKT and intestinal tissue regeneration. In conclusion, the PI3k activity of IPMK is necessary for PDK1-mediated AKT activation and intestinal homeostasis.
    Keywords:  Cell biology; Gastroenterology; Molecular biology
    DOI:  https://doi.org/10.1016/j.isci.2023.106623
  6. Dev Cell. 2023 May 16. pii: S1534-5807(23)00195-8. [Epub ahead of print]
    Actuation of single downstream nodes in growth factor network steers immune cell migration.
    Dhiman Sankar Pal, Tatsat Banerjee, Yiyan Lin, Félix de Trogoff, Jane Borleis, Pablo A Iglesias, Peter N Devreotes.
      Ras signaling is typically associated with cell growth, but not direct regulation of motility or polarity. By optogenetically targeting different nodes in the Ras/PI3K/Akt network in differentiated human HL-60 neutrophils, we abruptly altered protrusive activity, bypassing the chemoattractant receptor/G-protein network. First, global recruitment of active KRas4B/HRas isoforms or a RasGEF, RasGRP4, immediately increased spreading and random motility. Second, activating Ras at the cell rear generated new protrusions, reversed pre-existing polarity, and steered sustained migration in neutrophils or murine RAW 264.7 macrophages. Third, recruiting a RasGAP, RASAL3, to cell fronts extinguished protrusions and changed migration direction. Remarkably, persistent RASAL3 recruitment at stable fronts abrogated directed migration in three different chemoattractant gradients. Fourth, local recruitment of the Ras-mTORC2 effector, Akt, in neutrophils or Dictyostelium amoebae generated new protrusions and rearranged pre-existing polarity. Overall, these optogenetic effects were mTORC2-dependent but relatively independent of PI3K. Thus, receptor-independent, local activations of classical growth-control pathways directly control actin assembly, cell shape, and migration modes.
    Keywords:  actin cytoskeleton; biochemical excitability; cancer; development; immunity; immunotherapy; leukocytes; mTOR signaling; metastasis; optogenetics
    DOI:  https://doi.org/10.1016/j.devcel.2023.04.019
  7. Cell Rep. 2023 May 19. pii: S2211-1247(23)00547-8. [Epub ahead of print]42(5): 112536
    AKT activation because of PTEN loss upregulates xCT via GSK3β/NRF2, leading to inhibition of ferroptosis in PTEN-mutant tumor cells.
    Kaitlyn M Cahuzac, Abigail Lubin, Kaitlyn Bosch, Nicole Stokes, Sarah Mense Shoenfeld, Royce Zhou, Haddy Lemon, John Asara, Ramon E Parsons.
      Here, we show that the tumor suppressor phosphatase and tensin homolog deleted from chromosome 10 (PTEN) sensitizes cells to ferroptosis, an iron-dependent form of cell death, by restraining the expression and activity of the cystine/glutamate antiporter system Xc- (xCT). Loss of PTEN activates AKT kinase to inhibit GSK3β, increasing NF-E2 p45-related factor 2 (NRF2) along with transcription of one of its known target genes encoding xCT. Elevated xCT in Pten-null mouse embryonic fibroblasts increases the flux of cystine transport and synthesis of glutathione, which enhances the steady-state levels of these metabolites. A pan-cancer analysis finds that loss of PTEN shows evidence of increased xCT, and PTEN-mutant cells are resistant to ferroptosis as a consequence of elevated xCT. These findings suggest that selection of PTEN mutation during tumor development may be due to its ability to confer resistance to ferroptosis in the setting of metabolic and oxidative stress that occurs during tumor initiation and progression.
    Keywords:  Akt; CP: Cancer; CP: Metabolism; GSK3β; NRF2; PTEN; cancer; cysteine; ferroptosis; glutathione; xCT
    DOI:  https://doi.org/10.1016/j.celrep.2023.112536
  8. Front Immunol. 2023 ;14 1115244
    Dual inhibition of phosphoinositide 3-kinases delta and gamma reduces chronic B cell activation and autoantibody production in a mouse model of lupus.
    Folayemi Olayinka-Adefemi, Sen Hou, Aaron J Marshall.
      Phosphoinositide 3-kinase delta (PI3Kδ) plays key roles in normal B cell activation and is chronically activated in malignant B cells. Targeting of PI3Kδ using FDA-approved drugs Idelalisib or Umbralisib has shown efficacy in treatment of multiple B cell malignancies. Duvelisib, an inhibitor targeting both PI3Kδ and PI3Kγ (PI3Kδγi) has also been used for treatment of several leukemias and lymphomas and was suggested to offer potential additional benefits in supressing T cell and inflammatory responses. Transcriptomics analyses indicated that while most B cell subsets predominantly express PI3Kδ, plasma cells upregulate PI3Kγ. We thus assessed whether PI3Kδγi treatment can impact chronic B cell activation in the context of an autoantibody-mediated disease. Using the TAPP1R218LxTAPP2R211L (TAPP KI) mouse model of lupus-like disease driven by dysregulated PI3K pathway activity, we performed 4 week PI3Kδγi treatments and found significant reduction in CD86+ B cells, germinal center B cells, follicular helper T cells and plasma cells in multiple tissues. This treatment also significantly attenuated the abnormally elevated serum levels of IgG isotypes observed in this model. The profile of autoantibodies generated was markedly altered by PI3Kδγi treatment, with significant reductions in IgM and IgG targeting nuclear antigens, matrix proteins and other autoantigens. Kidney pathology was also impacted, with reduced IgG deposition and glomerulonephritis. These results indicate that dual inhibition of PI3Kδ and PI3Kγ can target autoreactive B cells and may have therapeutic benefits in autoantibody-mediated disease.
    Keywords:  B lymphocytes; PI3K signaling pathway; antibodies; autoimmunity; germinal center; plasma cell
    DOI:  https://doi.org/10.3389/fimmu.2023.1115244
  9. Proc Natl Acad Sci U S A. 2023 05 30. 120(22): e2219854120
    Mechanosensitive stem cell fate choice is instructed by dynamic fluctuations in activation of Rho GTPases.
    Rocío G Sampayo, Mason Sakamoto, Madeline Wang, Sanjay Kumar, David V Schaffer.
      During the intricate process by which cells give rise to tissues, embryonic and adult stem cells are exposed to diverse mechanical signals from the extracellular matrix (ECM) that influence their fate. Cells can sense these cues in part through dynamic generation of protrusions, modulated and controlled by cyclic activation of Rho GTPases. However, it remains unclear how extracellular mechanical signals regulate Rho GTPase activation dynamics and how such rapid, transient activation dynamics are integrated to yield long-term, irreversible cell fate decisions. Here, we report that ECM stiffness cues alter not only the magnitude but also the temporal frequency of RhoA and Cdc42 activation in adult neural stem cells (NSCs). Using optogenetics to control the frequency of RhoA and Cdc42 activation, we further demonstrate that these dynamics are functionally significant, where high- vs. low-frequency activation of RhoA and Cdc42 drives astrocytic vs. neuronal differentiation, respectively. In addition, high-frequency Rho GTPase activation induces sustained phosphorylation of the TGFβ pathway effector SMAD1, which in turn drives the astrocytic differentiation. By contrast, under low-frequency Rho GTPase stimulation, cells fail to accumulate SMAD1 phosphorylation and instead undergo neurogenesis. Our findings reveal the temporal patterning of Rho GTPase signaling and the resulting accumulation of an SMAD1 signal as a critical mechanism through which ECM stiffness cues regulate NSC fate.
    Keywords:  Rho GTPases; mechanobiology; mechanosensing; optogenetics; stem cells
    DOI:  https://doi.org/10.1073/pnas.2219854120
  10. bioRxiv. 2023 May 12. pii: 2023.05.09.540043. [Epub ahead of print]
    Feature selection for preserving biological trajectories in single-cell data.
    Jolene S Ranek, Wayne Stallaert, Justin Milner, Natalie Stanley, Jeremy E Purvis.
      Single-cell technologies can readily measure the expression of thousands of molecular features from individual cells undergoing dynamic biological processes, such as cellular differentiation, immune response, and disease progression. While examining cells along a computationally ordered pseudotime offers the potential to study how subtle changes in gene or protein expression impact cell fate decision-making, identifying characteristic features that drive continuous biological processes remains difficult to detect from unenriched and noisy single-cell data. Given that all profiled sources of feature variation contribute to the cell-to-cell distances that define an inferred cellular trajectory, including confounding sources of biological variation (e.g. cell cycle or metabolic state) or noisy and irrelevant features (e.g. measurements with low signal-to-noise ratio) can mask the underlying trajectory of study and hinder inference. Here, we present DELVE (dynamic selection of locally covarying features), an unsupervised feature selection method for identifying a representative subset of dynamically-expressed molecular features that recapitulates cellular trajectories. In contrast to previous work, DELVE uses a bottom-up approach to mitigate the effect of unwanted sources of variation confounding inference, and instead models cell states from dynamic feature modules that constitute core regulatory complexes. Using simulations, single-cell RNA sequencing data, and iterative immunofluorescence imaging data in the context of the cell cycle and cellular differentiation, we demonstrate that DELVE selects features that more accurately characterize cell populations and improve the recovery of cell type transitions. This feature selection framework provides an alternative approach for improving trajectory inference and uncovering co-variation amongst features along a biological trajectory. DELVE is implemented as an open-source python package and is publicly available at: https://github.com/jranek/delve .
    DOI:  https://doi.org/10.1101/2023.05.09.540043
  11. Nat Rev Methods Primers. 2022 ;pii: 9. [Epub ahead of print]2(1):
    High-content CRISPR screening.
    Christoph Bock, Paul Datlinger, Florence Chardon, Matthew A Coelho, Matthew B Dong, Keith A Lawson, Tian Lu, Laetitia Maroc, Thomas M Norman, Bicna Song, Geoff Stanley, Sidi Chen, Mathew Garnett, Wei Li, Jason Moffat, Lei S Qi, Rebecca S Shapiro, Jay Shendure, Jonathan S Weissman, Xiaowei Zhuang.
      CRISPR screens are a powerful source of biological discovery, enabling the unbiased interrogation of gene function in a wide range of applications and species. In pooled CRISPR screens, various genetically encoded perturbations are introduced into pools of cells. The targeted cells proliferate under a biological challenge such as cell competition, drug treatment or viral infection. Subsequently, the perturbation-induced effects are evaluated by sequencing-based counting of the guide RNAs that specify each perturbation. The typical results of such screens are ranked lists of genes that confer sensitivity or resistance to the biological challenge of interest. Contributing to the broad utility of CRISPR screens, adaptations of the core CRISPR technology make it possible to activate, silence or otherwise manipulate the target genes. Moreover, high-content read-outs such as single-cell RNA sequencing and spatial imaging help characterize screened cells with unprecedented detail. Dedicated software tools facilitate bioinformatic analysis and enhance reproducibility. CRISPR screening has unravelled various molecular mechanisms in basic biology, medical genetics, cancer research, immunology, infectious diseases, microbiology and other fields. This Primer describes the basic and advanced concepts of CRISPR screening and its application as a flexible and reliable method for biological discovery, biomedical research and drug development - with a special emphasis on high-content methods that make it possible to obtain detailed biological insights directly as part of the screen.
    DOI:  https://doi.org/10.1038/s43586-022-00098-7
  12. Nat Commun. 2023 May 23. 14(1): 2978
    Genetically encoded photocatalytic protein labeling enables spatially-resolved profiling of intracellular proteome.
    Fu Zheng, Xinyue Zhou, Peng Zou, Chenxin Yu.
      Mapping the subcellular organization of proteins is crucial for understanding their biological functions. Herein, we report a reactive oxygen species induced protein labeling and identification (RinID) method for profiling subcellular proteome in the context of living cells. Our method capitalizes on a genetically encoded photocatalyst, miniSOG, to locally generate singlet oxygen that reacts with proximal proteins. Labeled proteins are conjugated in situ with an exogenously supplied nucleophilic probe, which serves as a functional handle for subsequent affinity enrichment and mass spectrometry-based protein identification. From a panel of nucleophilic compounds, we identify biotin-conjugated aniline and propargyl amine as highly reactive probes. As a demonstration of the spatial specificity and depth of coverage in mammalian cells, we apply RinID in the mitochondrial matrix, capturing 477 mitochondrial proteins with 94% specificity. We further demonstrate the broad applicability of RinID in various subcellular compartments, including the nucleus and the endoplasmic reticulum (ER). The temporal control of RinID enables pulse-chase labeling of ER proteome in HeLa cells, which reveals substantially higher clearance rate for secreted proteins than ER resident proteins.
    DOI:  https://doi.org/10.1038/s41467-023-38565-8
  13. J Proteome Res. 2023 May 23.
    LFQ-Based Peptide and Protein Intensity Differential Expression Analysis.
    Mingze Bai, Jingwen Deng, Chengxin Dai, Julianus Pfeuffer, Timo Sachsenberg, Yasset Perez-Riverol.
      Testing for significant differences in quantities at the protein level is a common goal of many LFQ-based mass spectrometry proteomics experiments. Starting from a table of protein and/or peptide quantities from a given proteomics quantification software, many tools and R packages exist to perform the final tasks of imputation, summarization, normalization, and statistical testing. To evaluate the effects of packages and settings in their substeps on the final list of significant proteins, we studied several packages on three public data sets with known expected protein fold changes. We found that the results between packages and even across different parameters of the same package can vary significantly. In addition to usability aspects and feature/compatibility lists of different packages, this paper highlights sensitivity and specificity trade-offs that come with specific packages and settings.
    Keywords:  data analysis; imputation and normalization algorithms; mass spectrometry proteomics; protein expression; protein fold changes
    DOI:  https://doi.org/10.1021/acs.jproteome.2c00812
  14. Nat Biotechnol. 2023 May 25.
    Stabilized mosaic single-cell data integration using unshared features.
    Shila Ghazanfar, Carolina Guibentif, John C Marioni.
      Currently available single-cell omics technologies capture many unique features with different biological information content. Data integration aims to place cells, captured with different technologies, onto a common embedding to facilitate downstream analytical tasks. Current horizontal data integration techniques use a set of common features, thereby ignoring non-overlapping features and losing information. Here we introduce StabMap, a mosaic data integration technique that stabilizes mapping of single-cell data by exploiting the non-overlapping features. StabMap first infers a mosaic data topology based on shared features, then projects all cells onto supervised or unsupervised reference coordinates by traversing shortest paths along the topology. We show that StabMap performs well in various simulation contexts, facilitates 'multi-hop' mosaic data integration where some datasets do not share any features and enables the use of spatial gene expression features for mapping dissociated single-cell data onto a spatial transcriptomic reference.
    DOI:  https://doi.org/10.1038/s41587-023-01766-z
  15. Mol Ther Nucleic Acids. 2023 Jun 13. 32 536-552
    Strategies for precise gene edits in mammalian cells.
    Katye M Fichter, Tahereh Setayesh, Punam Malik.
      CRISPR-Cas technologies have the potential to revolutionize genetic medicine. However, work is still needed to make this technology clinically efficient for gene correction. A barrier to making precise genetic edits in the human genome is controlling how CRISPR-Cas-induced DNA breaks are repaired by the cell. Since error-prone non-homologous end-joining is often the preferred cellular repair pathway, CRISPR-Cas-induced breaks often result in gene disruption. Homology-directed repair (HDR) makes precise genetic changes and is the clinically desired pathway, but this repair pathway requires a homology donor template and cycling cells. Newer editing strategies, such as base and prime editing, can affect precise repair for relatively small edits without requiring HDR and circumvent cell cycle dependence. However, these technologies have limitations in the extent of genetic editing and require the delivery of bulky cargo. Here, we discuss the pros and cons of precise gene correction using CRISPR-Cas-induced HDR, as well as base and prime editing for repairing small mutations. Finally, we consider emerging new technologies, such as recombination and transposases, which can circumvent both cell cycle and cellular DNA repair dependence for editing the genome.
    Keywords:  CRISPR-Cas9; MT: RNA/DNA Editing; base editing; gene editing; homology-directed repair; prime editing
    DOI:  https://doi.org/10.1016/j.omtn.2023.04.012
  16. Proc Natl Acad Sci U S A. 2023 05 30. 120(22): e2221127120
    Efficient precise integration of large DNA sequences with 3'-overhang dsDNA donors using CRISPR/Cas9.
    Wenjie Han, Zhigang Li, Yijun Guo, Kaining He, Wenqing Li, Caoling Xu, Lishuang Ge, Miao He, Xue Yin, Junxiang Zhou, Chengxu Li, Dongbao Yao, Jianqiang Bao, Haojun Liang.
      CRISPR/Cas9 genome-editing tools have tremendously boosted our capability of manipulating the eukaryotic genomes in biomedical research and innovative biotechnologies. However, the current approaches that allow precise integration of gene-sized large DNA fragments generally suffer from low efficiency and high cost. Herein, we developed a versatile and efficient approach, termed LOCK (Long dsDNA with 3'-Overhangs mediated CRISPR Knock-in), by utilizing specially designed 3'-overhang double-stranded DNA (odsDNA) donors harboring 50-nt homology arm. The length of the 3'-overhangs of odsDNA is specified by the five consecutive phosphorothioate modifications. Compared with existing methods, LOCK allows highly efficient targeted insertion of kilobase-sized DNA fragments into the mammalian genomes with low cost and low off-target effects, yielding >fivefold higher knock-in frequencies than conventional homologous recombination-based approaches. This newly designed LOCK approach based on homology-directed repair is a powerful tool suitable for gene-sized fragment integration that is urgently needed for genetic engineering, gene therapies, and synthetic biology.
    Keywords:  3′-overhangs dsDNA; CRISPR knock-in; double-stranded break; off-target effect; phosphorothioate modification
    DOI:  https://doi.org/10.1073/pnas.2221127120
  17. PLoS Comput Biol. 2023 May 22. 19(5): e1011135
    Filter inference: A scalable nonlinear mixed effects inference approach for snapshot time series data.
    David Augustin, Ben Lambert, Ken Wang, Antje-Christine Walz, Martin Robinson, David Gavaghan.
      Variability is an intrinsic property of biological systems and is often at the heart of their complex behaviour. Examples range from cell-to-cell variability in cell signalling pathways to variability in the response to treatment across patients. A popular approach to model and understand this variability is nonlinear mixed effects (NLME) modelling. However, estimating the parameters of NLME models from measurements quickly becomes computationally expensive as the number of measured individuals grows, making NLME inference intractable for datasets with thousands of measured individuals. This shortcoming is particularly limiting for snapshot datasets, common e.g. in cell biology, where high-throughput measurement techniques provide large numbers of single cell measurements. We introduce a novel approach for the estimation of NLME model parameters from snapshot measurements, which we call filter inference. Filter inference uses measurements of simulated individuals to define an approximate likelihood for the model parameters, avoiding the computational limitations of traditional NLME inference approaches and making efficient inferences from snapshot measurements possible. Filter inference also scales well with the number of model parameters, using state-of-the-art gradient-based MCMC algorithms such as the No-U-Turn Sampler (NUTS). We demonstrate the properties of filter inference using examples from early cancer growth modelling and from epidermal growth factor signalling pathway modelling.
    DOI:  https://doi.org/10.1371/journal.pcbi.1011135
  18. Nat Biotechnol. 2023 May 25.
    Dictionary learning for integrative, multimodal and scalable single-cell analysis.
    Yuhan Hao, Tim Stuart, Madeline H Kowalski, Saket Choudhary, Paul Hoffman, Austin Hartman, Avi Srivastava, Gesmira Molla, Shaista Madad, Carlos Fernandez-Granda, Rahul Satija.
      Mapping single-cell sequencing profiles to comprehensive reference datasets provides a powerful alternative to unsupervised analysis. However, most reference datasets are constructed from single-cell RNA-sequencing data and cannot be used to annotate datasets that do not measure gene expression. Here we introduce 'bridge integration', a method to integrate single-cell datasets across modalities using a multiomic dataset as a molecular bridge. Each cell in the multiomic dataset constitutes an element in a 'dictionary', which is used to reconstruct unimodal datasets and transform them into a shared space. Our procedure accurately integrates transcriptomic data with independent single-cell measurements of chromatin accessibility, histone modifications, DNA methylation and protein levels. Moreover, we demonstrate how dictionary learning can be combined with sketching techniques to improve computational scalability and harmonize 8.6 million human immune cell profiles from sequencing and mass cytometry experiments. Our approach, implemented in version 5 of our Seurat toolkit ( http://www.satijalab.org/seurat ), broadens the utility of single-cell reference datasets and facilitates comparisons across diverse molecular modalities.
    DOI:  https://doi.org/10.1038/s41587-023-01767-y
  19. Aging Cell. 2023 May 24. e13888
    Unbiased evaluation of rapamycin's specificity as an mTOR inhibitor.
    Filippo Artoni, Nina Grützmacher, Constantinos Demetriades.
      Rapamycin is a macrolide antibiotic that functions as an immunosuppressive and anti-cancer agent, and displays robust anti-ageing effects in multiple organisms including humans. Importantly, rapamycin analogues (rapalogs) are of clinical importance against certain cancer types and neurodevelopmental diseases. Although rapamycin is widely perceived as an allosteric inhibitor of mTOR (mechanistic target of rapamycin), the master regulator of cellular and organismal physiology, its specificity has not been thoroughly evaluated so far. In fact, previous studies in cells and in mice hinted that rapamycin may be also acting independently from mTOR to influence various cellular processes. Here, we generated a gene-edited cell line that expresses a rapamycin-resistant mTOR mutant (mTORRR ) and assessed the effects of rapamycin treatment on the transcriptome and proteome of control or mTORRR -expressing cells. Our data reveal a striking specificity of rapamycin towards mTOR, demonstrated by virtually no changes in mRNA or protein levels in rapamycin-treated mTORRR cells, even following prolonged drug treatment. Overall, this study provides the first unbiased and conclusive assessment of rapamycin's specificity, with potential implications for ageing research and human therapeutics.
    Keywords:  ageing; mTORC1; proteomics; rapamycin; sirolimus
    DOI:  https://doi.org/10.1111/acel.13888
  20. bioRxiv. 2023 May 11. pii: 2023.05.11.539703. [Epub ahead of print]
    Discovery and Validation of Context-Dependent Synthetic Mammalian Promoters.
    Adam M Zahm, William S Owens, Samuel R Himes, Kathleen E Rondem, Braden S Fallon, Alexa N Gormick, Joshua S Bloom, Sriram Kosuri, Henry Chan, Justin G English.
      Cellular transcription enables cells to adapt to various stimuli and maintain homeostasis. Transcription factors bind to transcription response elements (TREs) in gene promoters, initiating transcription. Synthetic promoters, derived from natural TREs, can be engineered to control exogenous gene expression using endogenous transcription machinery. This technology has found extensive use in biological research for applications including reporter gene assays, biomarker development, and programming synthetic circuits in living cells. However, a reliable and precise method for selecting minimally-sized synthetic promoters with desired background, amplitude, and stimulation response profiles has been elusive. In this study, we introduce a massively parallel reporter assay library containing 6184 synthetic promoters, each less than 250 bp in length. This comprehensive library allows for rapid identification of promoters with optimal transcriptional output parameters across multiple cell lines and stimuli. We showcase this library's utility to identify promoters activated in unique cell types, and in response to metabolites, mitogens, cellular toxins, and agonism of both aminergic and non-aminergic GPCRs. We further show these promoters can be used in luciferase reporter assays, eliciting 50-100 fold dynamic ranges in response to stimuli. Our platform is effective, easily implemented, and provides a solution for selecting short-length promoters with precise performance for a multitude of applications.
    DOI:  https://doi.org/10.1101/2023.05.11.539703
  21. HGG Adv. 2023 Jul 13. 4(3): 100199
    The mitochondrial genome as a modifier of autism versus cancer phenotypes in PTEN hamartoma tumor syndrome.
    Ruipeng Wei, Lamis Yehia, Ying Ni, Charis Eng.
      Cancer and autism spectrum disorder/developmental delay (ASD/DD) are two common clinical phenotypes in individuals with germline PTEN variants (PTEN hamartoma tumor syndrome, PHTS). Burgeoning studies have shown that genomic and metabolomic factors may act as modifiers of ASD/DD versus cancer in PHTS. Recently, we showed copy number variations to be associated with ASD/DD versus cancer in these PHTS individuals. We also found that mitochondrial complex II variants occurring in 10% of PHTS individuals modify breast cancer risk and thyroid cancer histology. These studies suggest that mitochondrial pathways could act as important factors in PHTS phenotype development. However, the mitochondrial genome (mtDNA) has never been systematically studied in PHTS. We therefore investigated the mtDNA landscape extracted from whole-genome sequencing data from 498 PHTS individuals, including 164 with ASD/DD (PHTS-onlyASD/DD), 184 with cancer (PHTS-onlyCancer), 132 with neither ASD/DD nor cancer (PHTS-neither), and 18 with both ASD/DD and cancer (PHTS-ASDCancer). We demonstrate that PHTS-onlyASD/DD has significantly higher mtDNA copy number than PHTS-onlyCancer group (p = 9.2 × 10-3 in all samples; p = 4.2 × 10-3 in the H haplogroup). PHTS-neither group has significantly higher mtDNA variant burden than PHTS-ASDCancer group (p = 4.6 × 10-2); the PHTS-noCancer group (PHTS-onlyASD/DD and PHTS-neither groups) also shows higher variant burden than the PHTS-Cancer group (PHTS-onlyCancer and PHTS-ASD/Cancer groups; p = 3.3 × 10-2). Our study implicates the mtDNA as a modifier of ASD/DD versus cancer phenotype development in PHTS.
    Keywords:  PTEN; autism spectrum disorder; cancer; genomic modifiers; mitochondrial DNA (mtDNA)
    DOI:  https://doi.org/10.1016/j.xhgg.2023.100199
  22. Curr Opin Chem Biol. 2023 May 22. pii: S1367-5931(23)00065-0. [Epub ahead of print]75 102327
    Spatial single cell metabolomics: Current challenges and future developments.
    Kyle D G Saunders, Holly-May Lewis, Dany Jv Beste, Olivier Cexus, Melanie J Bailey.
      Single cell metabolomics is a rapidly advancing field of bio-analytical chemistry which aims to observe cellular biology with the greatest detail possible. Mass spectrometry imaging and selective cell sampling (e.g. using nanocapillaries) are two common approaches within the field. Recent achievements such as observation of cell-cell interactions, lipids determining cell states and rapid phenotypic identification demonstrate the efficacy of these approaches and the momentum of the field. However, single cell metabolomics can only continue with the same impetus if the universal challenges to the field are met, such as the lack of strategies for standardisation and quantification, and lack of specificity/sensitivity. Mass spectrometry imaging and selective cell sampling come with unique advantages and challenges which, in many cases are complementary to each other. We propose here that the challenges specific to each approach could be ameliorated with collaboration between the two communities driving these approaches.
    Keywords:  Lipidomics; Mass spectrometry; Mass spectrometry imaging; Selective cell sampling; Single cell metabolomics
    DOI:  https://doi.org/10.1016/j.cbpa.2023.102327
  23. iScience. 2023 Jun 16. 26(6): 106767
    Nearly maximal information gain due to time integration in central dogma reactions.
    Swarnavo Sarkar, Jayan Rammohan.
      Living cells process information about their environment through the central dogma processes of transcription and translation, which drive the cellular response to stimuli. Here, we study the transfer of information from environmental input to the transcript and protein expression levels. Evaluation of both experimental and analogous simulation data reveals that transcription and translation are not two simple information channels connected in series. Instead, we demonstrate that the central dogma reactions often create a time-integrating information channel, where the translation channel receives and integrates multiple outputs from the transcription channel. This information channel model of the central dogma provides new information-theoretic selection criteria for the central dogma rate constants. Using the data for four well-studied species we show that their central dogma rate constants achieve information gain because of time integration while also keeping the loss because of stochasticity in translation relatively low (<0.5 bits).
    Keywords:  Biophysics; Gene process; Information system model
    DOI:  https://doi.org/10.1016/j.isci.2023.106767
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