bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2023‒03‒05
29 papers selected by
Ralitsa Radostinova Madsen
University College London Cancer Institute
  1. Molecular basis for differential activation of p101 and p84 complexes of PI3Kγ by Ras and GPCRs.
  2. Tumour suppressor PTEN activity is differentially inducible by myo-inositol phosphates.
  3. An open label trial of nemiralisib, an inhaled PI3 kinase delta inhibitor for the treatment of Activated PI3 kinase Delta Syndrome.
  4. Simultaneous Inhibition of PI3Kgamma and PI3Kdelta Deteriorates T-cell Function With Implications for Chronic Lymphocytic Leukemia.
  5. Blebs promote cell survival by assembling oncogenic signalling hubs.
  6. Efficient bi-allelic tagging in human induced pluripotent stem cells using CRISPR.
  7. Ras protein abundance correlates with Ras isoform mutation patterns in cancer.
  8. PIK3CA copy number gain and inhibitors of the PI3K/AKT/mTOR pathway in triple-negative breast cancer.
  9. Frequent PIK3CA mutation in normal endometrial gland drives spheroid formation and may be involved in stem cell propagation.
  10. What is cancer metabolism?
  11. Single-cell proteomics enabled by next-generation sequencing or mass spectrometry.
  12. In silico tissue generation and power analysis for spatial omics.
  13. mTORC1 upregulates B7-H3/CD276 to inhibit antitumor T cells and drive tumor immune evasion.
  14. Injury primes mutation-bearing astrocytes for dedifferentiation in later life.
  15. scPrisma infers, filters and enhances topological signals in single-cell data using spectral template matching.
  16. Mechanosensitive mTORC2 independently coordinates leading and trailing edge polarity programs during neutrophil migration.
  17. State- and stimulus-specific dynamics of SMAD signaling determine fate decisions in individual cells.
  18. PI3king apart a rare disease with targeted therapy.
  19. Initial recommendations for performing, benchmarking and reporting single-cell proteomics experiments.
  20. SpliceTools, a suite of downstream RNA splicing analysis tools to investigate mechanisms and impact of alternative splicing.
  21. Interstrand crosslinking of homologous repair template DNA enhances gene editing in human cells.
  22. Bleb protrusions help cancer cells to cheat death.
  23. RNA splicing analysis using heterogeneous and large RNA-seq datasets.
  24. FAK-mediated phosphorylation at Y464 regulates p85β nuclear translocation to promote tumorigenesis of ccRCC by repressing RB1 expression.
  25. New advances in CRISPR/Cas-mediated precise gene-editing techniques.
  26. Single-Cell Molecular Barcoding to Decode Multimodal Information Defining Cell States.
  27. Integrative analysis of multimodal mass spectrometry data in MZmine 3.
  28. Lysosomal control of senescence and inflammation through cholesterol partitioning.
  29. Proximal binding of dCas9 at a DNA double strand break stimulates homology-directed repair as a local inhibitor of classical non-homologous end joining.
  1. Cell Rep. 2023 Feb 25. pii: S2211-1247(23)00183-3. [Epub ahead of print]42(3): 112172
    Molecular basis for differential activation of p101 and p84 complexes of PI3Kγ by Ras and GPCRs.
    Manoj K Rathinaswamy, Meredith L Jenkins, Benjamin R Duewell, Xuxiao Zhang, Noah J Harris, John T Evans, Jordan T B Stariha, Udit Dalwadi, Kaelin D Fleming, Harish Ranga-Prasad, Calvin K Yip, Roger L Williams, Scott D Hansen, John E Burke.
      Class IB phosphoinositide 3-kinase (PI3Kγ) is activated in immune cells and can form two distinct complexes (p110γ-p84 and p110γ-p101), which are differentially activated by G protein-coupled receptors (GPCRs) and Ras. Using a combination of X-ray crystallography, hydrogen deuterium exchange mass spectrometry (HDX-MS), electron microscopy, molecular modeling, single-molecule imaging, and activity assays, we identify molecular differences between p110γ-p84 and p110γ-p101 that explain their differential membrane recruitment and activation by Ras and GPCRs. The p110γ-p84 complex is dynamic compared with p110γ-p101. While p110γ-p101 is robustly recruited by Gβγ subunits, p110γ-p84 is weakly recruited to membranes by Gβγ subunits alone and requires recruitment by Ras to allow for Gβγ activation. We mapped two distinct Gβγ interfaces on p101 and the p110γ helical domain, with differences in the C-terminal domain of p84 and p101 conferring sensitivity of p110γ-p101 to Gβγ activation. Overall, our work provides key insight into the molecular basis for how PI3Kγ complexes are activated.
    Keywords:  CP: Cell biology; GPCR; HDX-MS; PI3K; PIK3CG; PIK3R5; PIK3R6; TIRF; p101; p84; phosphoinositide 3-kinase
    DOI:  https://doi.org/10.1016/j.celrep.2023.112172
  2. J Cell Mol Med. 2023 Feb 27.
    Tumour suppressor PTEN activity is differentially inducible by myo-inositol phosphates.
    Krzysztof Grzymajlo, Bouchra El Hafny-Rahbi, Claudine Kieda.
      Tumour evolution and efficacy of treatments are controlled by the microenvironment, the composition of which is primarily dependent on the angiogenic reaction to hypoxic stress. Tumour angiogenesis normalization is a challenge for adjuvant therapy strategies to chemo-, radio- and immunotherapeutics. Myo-inositol trispyrophosphate (ITPP) appears to provide the means to alleviate hypoxia in the tumour site by a double molecular mechanism. First, it modifies the properties of red blood cells (RBC) to release oxygen (O2 ) in the hypoxic sites more easily, leading to a rapid and stable increase in the partial pressure of oxygen (pO2 ). And second, it activates the endothelial phosphatase and tensin homologue deleted on Chromosome 10 (PTEN). The hypothesis that stable normalization of the vascular system is due to the PTEN, a tumour suppressor and phosphatase which controls the proper angiogenic reaction was ascertained. Here, by direct biochemical measurements of PTEN competitive activity in relation to PIP2 production, we show that the kinetics are complex in terms of the activation/inhibition effects of ITPP with an inverted consequence towards the kinase PI3K. The use of the surface plasmon resonance (SPR) technique allowed us to demonstrate that PTEN binds inositol derivatives differently but weakly. This method permitted us to reveal that PTEN is highly sensitive to the local concentration conditions, especially that ITPP increases the PTEN activity towards PIP3, and importantly, that PTEN affinity for ITPP is considerably increased by the presence of PIP3, as occurs in vivo. Our approach demonstrates the validity of using ITPP to activate PTEN for stable vessel normalization strategies.
    Keywords:  ITPP; PTEN; angiogenesis; cancer; hypoxia
    DOI:  https://doi.org/10.1111/jcmm.17699
  3. Pulm Pharmacol Ther. 2023 Feb 24. pii: S1094-5539(23)00013-5. [Epub ahead of print]79 102201
    An open label trial of nemiralisib, an inhaled PI3 kinase delta inhibitor for the treatment of Activated PI3 kinase Delta Syndrome.
    Malcolm Begg, Augustin Amour, Emily Jarvis, Teresa Tang, Sara Santos Franco, Andrew Want, Misba Beerahee, Disala Fernando, Yakshitha Karkera, Clare Sander, Thomas Southworth, Dave Singh, Jonathan Clark, Sergey Nejentsev, Klaus Okkenhaug, Alison Condliffe, Anita Chandra, Anthony Cahn, Edward Banham Hall.
      Activated PI3Kδ Syndrome (APDS) is a rare inherited inborn error of immunity caused by mutations that constitutively activate the p110 delta isoform of phosphoinositide 3-kinase (PI3Kδ), resulting in recurring pulmonary infections. Currently no licensed therapies are available. Here we report the results of an open-label trial in which five subjects were treated for 12 weeks with nemiralisib, an inhaled inhibitor of PI3Kδ, to determine safety, systemic exposure, together with lung and systemic biomarker profiles (Clinicaltrial.gov: NCT02593539). Induced sputum was captured to measure changes in phospholipids and inflammatory mediators, and blood samples were collected to assess pharmacokinetics of nemiralisib, and systemic biomarkers. Nemiralisib was shown to have an acceptable safety and tolerability profile, with cough being the most common adverse event, and no severe adverse events reported during the study. No meaningful changes in phosphatidylinositol (3,4,5)-trisphosphate (PIP3; the enzyme product of PI3Kδ) or downstream inflammatory markers in induced sputum, were observed following nemiralisib treatment. Similarly, there were no meaningful changes in blood inflammatory markers, or lymphocytes subsets. Systemic levels of nemiralisib were higher in subjects in this study compared to previous observations. While nemiralisib had an acceptable safety profile, there was no convincing evidence of target engagement in the lung following inhaled dosing and no downstream effects observed in either the lung or blood compartments. We speculate that this could be explained by nemiralisib not being retained in the lung for sufficient duration, suggested by the increased systemic exposure, perhaps due to pre-existing structural lung damage. In this study investigating a small number of subjects with APDS, nemiralisib appeared to be safe and well-tolerated. However, data from this study do not support the hypothesis that inhaled treatment with nemiralisib would benefit patients with APDS.
    Keywords:  APDS; Biomarkers; Immunology; Inhalation; Nemiralisib; PI3Kδ
    DOI:  https://doi.org/10.1016/j.pupt.2023.102201
  4. Hemasphere. 2023 Mar;7(3): e840
    Simultaneous Inhibition of PI3Kgamma and PI3Kdelta Deteriorates T-cell Function With Implications for Chronic Lymphocytic Leukemia.
    Sebastian Faehling, Mariana Coelho, Alessia Floerchinger, Christof Schneider, Stephan Stilgenbauer, Peter Lichter, Martina Seiffert, Philipp M Roessner.
      Chronic lymphocytic leukemia (CLL) is a common and incurable B-cell malignancy. Recent therapeutic approaches that target the B-cell receptor signaling pathway include inhibition of phosphatidylinositol-3-kinase (PI3K). The PI3K isoform delta is constitutively active in CLL, making it an attractive therapeutic target. However, the expression of PI3K isoforms is not exclusive to leukemic cells, as other immune cells in the tumor microenvironment also rely on PI3K activity. Subsequently, therapeutic inhibition of PI3K causes immune-related adverse events (irAEs). Here, we analyzed the impact of the clinically approved PI3Kδ inhibitors idelalisib and umbralisib, the PI3Kγ inhibitor eganelisib, and the dual-γ and -δ inhibitor duvelisib on the functional capacity of T cells. All investigated inhibitors reduced T-cell activation and proliferation in vitro, which is in line with PI3K being a crucial signaling component of the T-cell receptor signaling. Further, dual inhibition of PI3Kγ and PI3Kδ showed strong additive effects suggesting a role also for PI3Kγ in T cells. Extrapolation of this data to a clinical setting could provide an explanation for the observed irAEs in CLL patients undergoing treatment with PI3K inhibitors. Consequently, this highlights the need for a close monitoring of patients treated with PI3K inhibitors, and particularly duvelisib, due to their potentially increased risk of T-cell deficiencies and associated infections.
    DOI:  https://doi.org/10.1097/HS9.0000000000000840
  5. Nature. 2023 Mar 01.
    Blebs promote cell survival by assembling oncogenic signalling hubs.
    Andrew D Weems, Erik S Welf, Meghan K Driscoll, Felix Y Zhou, Hanieh Mazloom-Farsibaf, Bo-Jui Chang, Vasanth S Murali, Gabriel M Gihana, Byron G Weiss, Joseph Chi, Divya Rajendran, Kevin M Dean, Reto Fiolka, Gaudenz Danuser.
      Most human cells require anchorage for survival. Cell-substrate adhesion activates diverse signalling pathways, without which cells undergo anoikis-a form of programmed cell death1. Acquisition of anoikis resistance is a pivotal step in cancer disease progression, as metastasizing cells often lose firm attachment to surrounding tissue2,3. In these poorly attached states, cells adopt rounded morphologies and form small hemispherical plasma membrane protrusions called blebs4-11. Bleb function has been thoroughly investigated in the context of amoeboid migration, but it has been examined far less in other scenarios12. Here we show by three-dimensional imaging and manipulation of cell morphological states that blebbing triggers the formation of plasma membrane-proximal signalling hubs that confer anoikis resistance. Specifically, in melanoma cells, blebbing generates plasma membrane contours that recruit curvature-sensing septin proteins as scaffolds for constitutively active mutant NRAS and effectors. These signalling hubs activate ERK and PI3K-well-established promoters of pro-survival pathways. Inhibition of blebs or septins has little effect on the survival of well-adhered cells, but in detached cells it causes NRAS mislocalization, reduced MAPK and PI3K activity, and ultimately, death. This unveils a morphological requirement for mutant NRAS to operate as an effective oncoprotein. Furthermore, whereas some BRAF-mutated melanoma cells do not rely on this survival pathway in a basal state, inhibition of BRAF and MEK strongly sensitizes them to both bleb and septin inhibition. Moreover, fibroblasts engineered to sustain blebbing acquire the same anoikis resistance as cancer cells even without harbouring oncogenic mutations. Thus, blebs are potent signalling organelles capable of integrating myriad cellular information flows into concerted cellular responses, in this case granting robust anoikis resistance.
    DOI:  https://doi.org/10.1038/s41586-023-05758-6
  6. STAR Protoc. 2023 Feb 01. pii: S2666-1667(23)00042-4. [Epub ahead of print]4(1): 102084
    Efficient bi-allelic tagging in human induced pluripotent stem cells using CRISPR.
    Xingjie Ren, Maya Asami Takagi, Yin Shen.
      Allelic tagging of endogenous genes enables studying gene function and transcriptional control in the native genomic context. Here, we present an efficient protocol for bi-allelic tagging of protein-coding genes with fluorescent reporters in human iPSCs using the CRISPR-Cas9-mediated homology-directed repair. We detail steps for design, cloning, electroporation, and single-cell clone isolation and validation. The tagging strategy described in this protocol is readily applicable for knockin of other reporters in diverse cell types for biomedical research.
    Keywords:  CRISPR; Cell Culture; Genetics; Molecular Biology; Stem Cells
    DOI:  https://doi.org/10.1016/j.xpro.2023.102084
  7. Oncogene. 2023 Mar 02.
    Ras protein abundance correlates with Ras isoform mutation patterns in cancer.
    Fiona E Hood, Yasmina M Sahraoui, Rosalind E Jenkins, Ian A Prior.
      Activating mutations of Ras genes are often observed in cancer. The protein products of the three Ras genes are almost identical. However, for reasons that remain unclear, KRAS is far more frequently mutated than the other Ras isoforms in cancer and RASopathies. We have quantified HRAS, NRAS, KRAS4A and KRAS4B protein abundance across a large panel of cell lines and healthy tissues. We observe consistent patterns of KRAS > NRAS»HRAS protein expression in cells that correlate with the rank order of Ras mutation frequencies in cancer. Our data provide support for the model of a sweet-spot of Ras dosage mediating isoform-specific contributions to cancer and development. We suggest that in most cases, being the most abundant Ras isoform correlates with occupying the sweet-spot and that HRAS and NRAS expression is usually insufficient to promote oncogenesis when mutated. However, our results challenge the notion that rare codons mechanistically underpin the predominance of KRAS mutant cancers. Finally, direct measurement of mutant versus wildtype KRAS protein abundance revealed a frequent imbalance that may suggest additional non-gene duplication mechanisms for optimizing oncogenic Ras dosage.
    DOI:  https://doi.org/10.1038/s41388-023-02638-1
  8. Cold Spring Harb Mol Case Stud. 2023 Mar 02. pii: mcs.a006255. [Epub ahead of print]
    PIK3CA copy number gain and inhibitors of the PI3K/AKT/mTOR pathway in triple-negative breast cancer.
    Ottavia Amato, Laurence Buisseret, Geraldine Gebhart, Nicolas Plouznikoff, Denis Larsimont, Ahmad Awada, Martine Piccart, Philippe Aftimos.
      As wider insights are gained on the molecular landscape of triple-negative breast cancer (TNBC), novel targeted therapeutic strategies might become an option in this setting as well. Activating mutations of PIK3CA represent the second most common alteration in TNBC after the TP53 mutation, with a prevalence of about 10-15%. Considering the well-established predictive role of PIK3CA mutations for response to agents targeting the PI3K/AKT/mTOR pathway, several clinical trials are currently evaluating these drugs in patients with advanced TNBC. However, much less is known regarding the actionability of PIK3CA copy number gains, which represent a thoroughly common molecular alteration in TNBC, with a prevalence estimated at 6-20%, and are listed as "likely gain-of-function" alterations in the OncoKB database. In the present paper we describe two clinical cases in which patients harboring PIK3CA-amplified TNBC received a targeted treatment with the mTOR-inhibitor everolimus and the PI3K-inhibitor alpelisib, respectively, with evidence of disease response on 18F-FDG Positron-emission tomography (PET) imaging. Hence, we discuss the evidence presently available regarding a possible predictive value of PIK3CA amplification for response to targeted treatment strategies, suggesting that this molecular alteration might represent an intriguing biomarker in this sense. Considering that few of the currently active clinical trials assessing agents targeting the PI3K/AKT/mTOR pathway in TNBC select patients based on tumor molecular characterization, and none of these based on PIK3CA copy number status, we urge for the introduction of PIK3CA amplification as a criterion for patient selection in future clinical trials in this setting.
    Keywords:  Neoplasm of the breast
    DOI:  https://doi.org/10.1101/mcs.a006255
  9. Cancer Sci. 2023 Feb 26.
    Frequent PIK3CA mutation in normal endometrial gland drives spheroid formation and may be involved in stem cell propagation.
    Seiya Sato, Kentaro Nakayama, Kosuke Kanno, Razia Sultana, Masako Ishikawa, Tomoka Ishibashi, Hitomi Yamashita, Satoru Kyo.
      Recent studies reported the presence of oncogenic mutations in normal endometrial glands, but the biological significance remain unclear. The present study investigated the status of KRAS/PIK3CA driver mutations in normal endometrial glands as well as the spheroid derived from single glands. The normal endometria of surgically removed uteri (n = 3) were divided into 9 regions, and 40 endometrial single glands were isolated from each region. The DNAs of 10 glands in each region were extracted and subjected to Sanger sequencing for KRAS or PIK3CA driver mutations, while the remaining 30 glands were conferred to a long-term spheroid culture, followed by Sanger sequencing. Immunohistochemical analyses of stem cell (Axin2, ALDH1A1, SOX9) markers were performed for spheroids. Sanger sequencing successfully detected oncogenic mutations of KRAS or PIK3CA in a single gland. Twenty-five of the 270 glands (9.3%) had mutations in either KRAS or PIK3CA, and the mutation frequency in each endometrial region varied from 0-50%. The droplet digital PCR showed high mutation allele frequency (MAF) of PIK3CA mutation, suggestive of clonal expansion of mutated cells within a gland. Over 60% of the collected spheroids had PIK3CA mutations, while no KRAS mutations were detected. Immunohistochemically, spheroids were mainly composed of cells with stem cell marker expressions. High MAF of PIK3CA mutation in a single gland as well as frequent PIK3CA mutation in stem-cell rich spheroids that originated from a single gland suggest the role of PIK3CA mutation in stem cell propagation. This information may improve our understanding of endometrial physiology as well as stem cell-oriented endometrial regeneration and carcinogenesis.
    Keywords:  Axin2; KRAS; PIK3CA; normal endometrium; stem cell
    DOI:  https://doi.org/10.1111/cas.15767
  10. Cell. 2023 Feb 22. pii: S0092-8674(23)00097-1. [Epub ahead of print]
    What is cancer metabolism?
    Lydia W S Finley.
      The uptake and metabolism of nutrients support fundamental cellular process from bioenergetics to biomass production and cell fate regulation. While many studies of cell metabolism focus on cancer cells, the principles of metabolism elucidated in cancer cells apply to a wide range of mammalian cells. The goal of this review is to discuss how the field of cancer metabolism provides a framework for revealing principles of cell metabolism and for dissecting the metabolic networks that allow cells to meet their specific demands. Understanding context-specific metabolic preferences and liabilities will unlock new approaches to target cancer cells to improve patient care.
    DOI:  https://doi.org/10.1016/j.cell.2023.01.038
  11. Nat Methods. 2023 Mar 02.
    Single-cell proteomics enabled by next-generation sequencing or mass spectrometry.
    Hayley M Bennett, William Stephenson, Christopher M Rose, Spyros Darmanis.
      In the last decade, single-cell RNA sequencing routinely performed on large numbers of single cells has greatly advanced our understanding of the underlying heterogeneity of complex biological systems. Technological advances have also enabled protein measurements, further contributing to the elucidation of cell types and states present in complex tissues. Recently, there have been independent advances in mass spectrometric techniques bringing us one step closer to characterizing single-cell proteomes. Here we discuss the challenges of detecting proteins in single cells by both mass spectrometry and sequencing-based methods. We review the state of the art for these techniques and propose that there is a space for technological advancements and complementary approaches that maximize the advantages of both classes of technologies.
    DOI:  https://doi.org/10.1038/s41592-023-01791-5
  12. Nat Methods. 2023 Mar 02.
    In silico tissue generation and power analysis for spatial omics.
    Ethan A G Baker, Denis Schapiro, Bianca Dumitrascu, Sanja Vickovic, Aviv Regev.
      As spatially resolved multiplex profiling of RNA and proteins becomes more prominent, it is increasingly important to understand the statistical power available to test specific hypotheses when designing and interpreting such experiments. Ideally, it would be possible to create an oracle that predicts sampling requirements for generalized spatial experiments. However, the unknown number of relevant spatial features and the complexity of spatial data analysis make this challenging. Here, we enumerate multiple parameters of interest that should be considered in the design of a properly powered spatial omics study. We introduce a method for tunable in silico tissue (IST) generation and use it with spatial profiling data sets to construct an exploratory computational framework for spatial power analysis. Finally, we demonstrate that our framework can be applied across diverse spatial data modalities and tissues of interest. While we demonstrate ISTs in the context of spatial power analysis, these simulated tissues have other potential use cases, including spatial method benchmarking and optimization.
    DOI:  https://doi.org/10.1038/s41592-023-01766-6
  13. Nat Commun. 2023 Mar 03. 14(1): 1214
    mTORC1 upregulates B7-H3/CD276 to inhibit antitumor T cells and drive tumor immune evasion.
    Heng-Jia Liu, Heng Du, Damir Khabibullin, Mahsa Zarei, Kevin Wei, Gordon J Freeman, David J Kwiatkowski, Elizabeth P Henske.
      Identifying the mechanisms underlying the regulation of immune checkpoint molecules and the therapeutic impact of targeting them in cancer is critical. Here we show that high expression of the immune checkpoint B7-H3 (CD276) and high mTORC1 activity correlate with immunosuppressive phenotypes and worse clinical outcomes in 11,060 TCGA human tumors. We find that mTORC1 upregulates B7-H3 expression via direct phosphorylation of the transcription factor YY2 by p70 S6 kinase. Inhibition of B7-H3 suppresses mTORC1-hyperactive tumor growth via an immune-mediated mechanism involving increased T-cell activity and IFN-γ responses coupled with increased tumor cell expression of MHC-II. CITE-seq reveals strikingly increased cytotoxic CD38+CD39+CD4+ T cells in B7-H3-deficient tumors. In pan-human cancers, a high cytotoxic CD38+CD39+CD4+ T-cell gene signature correlates with better clinical prognosis. These results show that mTORC1-hyperactivity, present in many human tumors including tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM), drives B7-H3 expression leading to suppression of cytotoxic CD4+ T cells.
    DOI:  https://doi.org/10.1038/s41467-023-36881-7
  14. Curr Biol. 2023 Feb 17. pii: S0960-9822(23)00152-5. [Epub ahead of print]
    Injury primes mutation-bearing astrocytes for dedifferentiation in later life.
    Holly Simpson Ragdale, Melanie Clements, Wenhao Tang, Elitza Deltcheva, Catia Andreassi, Alvina G Lai, Wai Hoong Chang, Maria Pandrea, Ivan Andrew, Laurence Game, Imran Uddin, Michael Ellis, Tariq Enver, Antonella Riccio, Samuel Marguerat, Simona Parrinello.
      Despite their latent neurogenic potential, most normal parenchymal astrocytes fail to dedifferentiate to neural stem cells in response to injury. In contrast, aberrant lineage plasticity is a hallmark of gliomas, and this suggests that tumor suppressors may constrain astrocyte dedifferentiation. Here, we show that p53, one of the most commonly inactivated tumor suppressors in glioma, is a gatekeeper of astrocyte fate. In the context of stab-wound injury, p53 loss destabilized the identity of astrocytes, priming them to dedifferentiate in later life. This resulted from persistent and age-exacerbated neuroinflammation at the injury site and EGFR activation in periwound astrocytes. Mechanistically, dedifferentiation was driven by the synergistic upregulation of mTOR signaling downstream of p53 loss and EGFR, which reinstates stemness programs via increased translation of neurodevelopmental transcription factors. Thus, our findings suggest that first-hit mutations remove the barriers to injury-induced dedifferentiation by sensitizing somatic cells to inflammatory signals, with implications for tumorigenesis.
    Keywords:  EGFR signaling; aging; astrocytes; brain injury; dedifferentiation; mTOR signaling; neural stem cells; neuroinflammation; p53
    DOI:  https://doi.org/10.1016/j.cub.2023.02.013
  15. Nat Biotechnol. 2023 Feb 27.
    scPrisma infers, filters and enhances topological signals in single-cell data using spectral template matching.
    Jonathan Karin, Yonathan Bornfeld, Mor Nitzan.
      Single-cell RNA sequencing has been instrumental in uncovering cellular spatiotemporal context. This task is challenging as cells simultaneously encode multiple, potentially cross-interfering, biological signals. Here we propose scPrisma, a spectral computational method that uses topological priors to decouple, enhance and filter different classes of biological processes in single-cell data, such as periodic and linear signals. We apply scPrisma to the analysis of the cell cycle in HeLa cells, circadian rhythm and spatial zonation in liver lobules, diurnal cycle in Chlamydomonas and circadian rhythm in the suprachiasmatic nucleus in the brain. scPrisma can be used to distinguish mixed cellular populations by specific characteristics such as cell type and uncover regulatory networks and cell-cell interactions specific to predefined biological signals, such as the circadian rhythm. We show scPrisma's flexibility in incorporating prior knowledge, inference of topologically informative genes and generalization to additional diverse templates and systems. scPrisma can be used as a stand-alone workflow for signal analysis and as a prior step for downstream single-cell analysis.
    DOI:  https://doi.org/10.1038/s41587-023-01663-5
  16. Mol Biol Cell. 2023 Mar 01. mbcE22050191
    Mechanosensitive mTORC2 independently coordinates leading and trailing edge polarity programs during neutrophil migration.
    Suvrajit Saha, Jason P Town, Orion D Weiner.
      By acting both upstream and downstream of biochemical organizers of the cytoskeleton, physical forces function as central integrators of cell shape and movement. Here we use a combination of genetic, pharmacological, and optogenetic perturbations to probe the role of the conserved mechanosensitive mTORC2 programs in neutrophil polarity and motility. We find that the tension-based inhibition of leading edge signals (Rac, F-actin) that underlies protrusion competition is gated by the kinase-independent role of the complex, whereas the regulation of RhoA and Myosin II-based contractility at the trailing edge depend on mTORC2 kinase activity. mTORC2 is essential for spatial and temporal coordination of the front and back polarity programs for persistent migration under confinement. This mechanosensory pathway integrates multiple upstream signals, and we find that membrane stretch synergizes with biochemical co-input PIP3 to robustly amplify mTORC2 activation. Our results suggest that different signalling arms of mTORC2 regulate spatially and molecularly divergent cytoskeletal programs for efficient coordination of neutrophil shape and movement. [Media: see text] [Media: see text].
    DOI:  https://doi.org/10.1091/mbc.E22-05-0191
  17. Proc Natl Acad Sci U S A. 2023 Mar 07. 120(10): e2210891120
    State- and stimulus-specific dynamics of SMAD signaling determine fate decisions in individual cells.
    Stefan Bohn, Lorenz Hexemer, Zixin Huang, Laura Strohmaier, Sonja Lenhardt, Stefan Legewie, Alexander Loewer.
      SMAD-mediated signaling regulates apoptosis, cell cycle arrest, and epithelial-to-mesenchymal transition to safeguard tissue homeostasis. However, it remains elusive how the relatively simple pathway can determine such a broad range of cell fate decisions and how it differentiates between varying ligands. Here, we systematically investigate how SMAD-mediated responses are modulated by various ligands of the transforming growth factor β (TGFβ) family and compare these ligand responses in quiescent and proliferating MCF10A cells. We find that the nature of the phenotypic response is mainly determined by the proliferation status, with migration and cell cycle arrest being dominant in proliferating cells for all tested TGFβ family ligands, whereas cell death is the major outcome in quiescent cells. In both quiescent and proliferating cells, the identity of the ligand modulates the strength of the phenotypic response proportional to the dynamics of induced SMAD nuclear-to-cytoplasmic translocation and, as a consequence, the corresponding gene expression changes. Interestingly, the proliferation state of a cell has little impact on the set of genes induced by SMAD signaling; instead, it modulates the relative cellular sensitivity to TGFβ superfamily members. Taken together, diversity of SMAD-mediated responses is mediated by differing cellular states, which determine ligand sensitivity and phenotypic effects, while the pathway itself merely serves as a quantitative relay from the cell membrane to the nucleus.
    Keywords:  SMAD; TGFβ superfamily; dynamics; signal transduction; single-cell analysis
    DOI:  https://doi.org/10.1073/pnas.2210891120
  18. Blood. 2023 Mar 02. 141(9): 963-964
    PI3king apart a rare disease with targeted therapy.
    Haley Newman, David T Teachey.
      
    DOI:  https://doi.org/10.1182/blood.2022019105
  19. Nat Methods. 2023 Mar 02.
    Initial recommendations for performing, benchmarking and reporting single-cell proteomics experiments.
    Laurent Gatto, Ruedi Aebersold, Juergen Cox, Vadim Demichev, Jason Derks, Edward Emmott, Alexander M Franks, Alexander R Ivanov, Ryan T Kelly, Luke Khoury, Andrew Leduc, Michael J MacCoss, Peter Nemes, David H Perlman, Aleksandra A Petelski, Christopher M Rose, Erwin M Schoof, Jennifer Van Eyk, Christophe Vanderaa, John R Yates, Nikolai Slavov.
      Analyzing proteins from single cells by tandem mass spectrometry (MS) has recently become technically feasible. While such analysis has the potential to accurately quantify thousands of proteins across thousands of single cells, the accuracy and reproducibility of the results may be undermined by numerous factors affecting experimental design, sample preparation, data acquisition and data analysis. We expect that broadly accepted community guidelines and standardized metrics will enhance rigor, data quality and alignment between laboratories. Here we propose best practices, quality controls and data-reporting recommendations to assist in the broad adoption of reliable quantitative workflows for single-cell proteomics. Resources and discussion forums are available at https://single-cell.net/guidelines .
    DOI:  https://doi.org/10.1038/s41592-023-01785-3
  20. Nucleic Acids Res. 2023 Mar 02. pii: gkad111. [Epub ahead of print]
    SpliceTools, a suite of downstream RNA splicing analysis tools to investigate mechanisms and impact of alternative splicing.
    Erik K Flemington, Samuel A Flemington, Tina M O'Grady, Melody Baddoo, Trang Nguyen, Yan Dong, Nathan A Ungerleider.
      As a fundamental aspect of normal cell signaling and disease states, there is great interest in determining alternative splicing (AS) changes in physiologic, pathologic, and pharmacologic settings. High throughput RNA sequencing and specialized software to detect AS has greatly enhanced our ability to determine transcriptome-wide splicing changes. Despite the richness of this data, deriving meaning from sometimes thousands of AS events is a substantial bottleneck for most investigators. We present SpliceTools, a suite of data processing modules that arms investigators with the ability to quickly produce summary statistics, mechanistic insights, and functional significance of AS changes through command line or through an online user interface. Utilizing RNA-seq datasets for 186 RNA binding protein knockdowns, nonsense mediated RNA decay inhibition, and pharmacologic splicing inhibition, we illustrate the utility of SpliceTools to distinguish splicing disruption from regulated transcript isoform changes, we show the broad transcriptome footprint of the pharmacologic splicing inhibitor, indisulam, we illustrate the utility in uncovering mechanistic underpinnings of splicing inhibition, we identify predicted neo-epitopes in pharmacologic splicing inhibition, and we show the impact of splicing alterations induced by indisulam on cell cycle progression. Together, SpliceTools puts rapid and easy downstream analysis at the fingertips of any investigator studying AS.
    DOI:  https://doi.org/10.1093/nar/gkad111
  21. Nat Biotechnol. 2023 Feb 27.
    Interstrand crosslinking of homologous repair template DNA enhances gene editing in human cells.
    Hannah I Ghasemi, Julien Bacal, Amanda C Yoon, Katherine U Tavasoli, Carmen Cruz, Jonathan T Vu, Brooke M Gardner, Chris D Richardson.
      We describe a strategy to boost the efficiency of gene editing via homology-directed repair (HDR) by covalently modifying the template DNA with interstrand crosslinks. Crosslinked templates (xHDRTs) increase Cas9-mediated editing efficiencies by up to fivefold in K562, HEK293T, U2OS, iPS and primary T cells. Increased editing from xHDRTs is driven by events on the template molecule and requires ataxia telangiectasia and Rad3-related (ATR) kinase and components of the Fanconi anemia pathway.
    DOI:  https://doi.org/10.1038/s41587-022-01654-y
  22. Nature. 2023 Mar 01.
    Bleb protrusions help cancer cells to cheat death.
    Michal Reichman-Fried, Erez Raz.
      
    Keywords:  Cancer; Cell biology
    DOI:  https://doi.org/10.1038/d41586-023-00477-4
  23. Nat Commun. 2023 Mar 03. 14(1): 1230
    RNA splicing analysis using heterogeneous and large RNA-seq datasets.
    Jorge Vaquero-Garcia, Joseph K Aicher, San Jewell, Matthew R Gazzara, Caleb M Radens, Anupama Jha, Scott S Norton, Nicholas F Lahens, Gregory R Grant, Yoseph Barash.
      The ubiquity of RNA-seq has led to many methods that use RNA-seq data to analyze variations in RNA splicing. However, available methods are not well suited for handling heterogeneous and large datasets. Such datasets scale to thousands of samples across dozens of experimental conditions, exhibit increased variability compared to biological replicates, and involve thousands of unannotated splice variants resulting in increased transcriptome complexity. We describe here a suite of algorithms and tools implemented in the MAJIQ v2 package to address challenges in detection, quantification, and visualization of splicing variations from such datasets. Using both large scale synthetic data and GTEx v8 as benchmark datasets, we assess the advantages of MAJIQ v2 compared to existing methods. We then apply MAJIQ v2 package to analyze differential splicing across 2,335 samples from 13 brain subregions, demonstrating its ability to offer insights into brain subregion-specific splicing regulation.
    DOI:  https://doi.org/10.1038/s41467-023-36585-y
  24. Cell Rep. 2023 Feb 28. pii: S2211-1247(23)00199-7. [Epub ahead of print]42(3): 112188
    FAK-mediated phosphorylation at Y464 regulates p85β nuclear translocation to promote tumorigenesis of ccRCC by repressing RB1 expression.
    Yanhua Zhang, Baoyu He, Dong Zhang, Yifan Zhang, Chengkun Chen, Wenye Zhang, Shiyi Yang, Meilian Yao, Gaoping Cui, Jun Gu, Ting Wang, Zhang Lin, Youben Fan, Zuquan Xiong, Yujun Hao.
      PI3K regulatory subunit p85s normally stabilizes and regulates catalytic subunit p110s in the cytoplasm. Recent studies show that p110-free p85s in the nucleus plays important roles in biological processes. However, the mechanisms by which p85s translocate into the nucleus remain elusive. Here, we describe the mechanism by which p85β translocates into the nucleus to promote ccRCC tumorigenesis. Phosphorylation of p85β at the Y464 by FAK facilitates its nuclear translocation in the kidney through enhancing the binding of p85β to KPNA1. PIK3R2/p85β is highly expressed in ccRCC samples and associated with overall survival of ccRCC patients. Nuclear but not cytoplasmic p85β performs oncogenic functions by repressing RB1 expression and regulating the G1/S cell cycle transition. Nuclear p85β represses RB1 expression by stabilizing histone methyltransferase EZH1/EZH2 proteins. Last, the FAK inhibitor defactinib significantly suppresses the tumor growth of ccRCC with high p85β Y464 levels.
    Keywords:  CP: Cancer; FAK; RB1; ccRCC; nuclear translocation; p85β; tyrosine phosphorylation
    DOI:  https://doi.org/10.1016/j.celrep.2023.112188
  25. Dis Model Mech. 2023 Feb 01. pii: dmm049874. [Epub ahead of print]16(2):
    New advances in CRISPR/Cas-mediated precise gene-editing techniques.
    Chris Richardson, Robert N Kelsh, Rebecca J Richardson.
      Over the past decade, CRISPR/Cas-based gene editing has become a powerful tool for generating mutations in a variety of model organisms, from Escherichia coli to zebrafish, rodents and large mammals. CRISPR/Cas-based gene editing effectively generates insertions or deletions (indels), which allow for rapid gene disruption. However, a large proportion of human genetic diseases are caused by single-base-pair substitutions, which result in more subtle alterations to protein function, and which require more complex and precise editing to recreate in model systems. Precise genome editing (PGE) methods, however, typically have efficiencies of less than a tenth of those that generate less-specific indels, and so there has been a great deal of effort to improve PGE efficiency. Such optimisations include optimal guide RNA and mutation-bearing donor DNA template design, modulation of DNA repair pathways that underpin how edits result from Cas-induced cuts, and the development of Cas9 fusion proteins that introduce edits via alternative mechanisms. In this Review, we provide an overview of the recent progress in optimising PGE methods and their potential for generating models of human genetic disease.
    Keywords:  Base/prime editing; CRISPR/Cas; HDR; Human disease modelling; Precise genome editing
    DOI:  https://doi.org/10.1242/dmm.049874
  26. Mol Cells. 2023 Feb 28. 46(2): 74-85
    Single-Cell Molecular Barcoding to Decode Multimodal Information Defining Cell States.
    Ik Soo Kim.
      Single-cell research has provided a breakthrough in biology to understand heterogeneous cell groups, such as tissues and organs, in development and disease. Molecular barcoding and subsequent sequencing technology insert a singlecell barcode into isolated single cells, allowing separation cell by cell. Given that multimodal information from a cell defines precise cellular states, recent technical advances in methods focus on simultaneously extracting multimodal data recorded in different biological materials (DNA, RNA, protein, etc.). This review summarizes recently developed singlecell multiomics approaches regarding genome, epigenome, and protein profiles with the transcriptome. In particular, we focus on how to anchor or tag molecules from a cell, improve throughputs with sample multiplexing, and record lineages, and we further discuss the future developments of the technology.
    Keywords:  molecular barcoding; multimodality; multiomics; single cell
    DOI:  https://doi.org/10.14348/molcells.2023.2168
  27. Nat Biotechnol. 2023 Mar 01.
    Integrative analysis of multimodal mass spectrometry data in MZmine 3.
    Robin Schmid, Steffen Heuckeroth, Ansgar Korf, Aleksandr Smirnov, Owen Myers, Thomas S Dyrlund, Roman Bushuiev, Kevin J Murray, Nils Hoffmann, Miaoshan Lu, Abinesh Sarvepalli, Zheng Zhang, Markus Fleischauer, Kai Dührkop, Mark Wesner, Shawn J Hoogstra, Edward Rudt, Olena Mokshyna, Corinna Brungs, Kirill Ponomarov, Lana Mutabdžija, Tito Damiani, Chris J Pudney, Mark Earll, Patrick O Helmer, Timothy R Fallon, Tobias Schulze, Albert Rivas-Ubach, Aivett Bilbao, Henning Richter, Louis-Félix Nothias, Mingxun Wang, Matej Orešič, Jing-Ke Weng, Sebastian Böcker, Astrid Jeibmann, Heiko Hayen, Uwe Karst, Pieter C Dorrestein, Daniel Petras, Xiuxia Du, Tomáš Pluskal.
      
    DOI:  https://doi.org/10.1038/s41587-023-01690-2
  28. Nat Metab. 2023 Mar 02.
    Lysosomal control of senescence and inflammation through cholesterol partitioning.
    Kyeonghwan Roh, Jeonghwan Noh, Yeonju Kim, Yeji Jang, Jaejin Kim, Haebeen Choi, Yeonghyeon Lee, Moongi Ji, Donghyun Kang, Mi-Sung Kim, Man-Jeong Paik, Jongkyeong Chung, Jin-Hong Kim, Chanhee Kang.
      Whereas cholesterol is vital for cell growth, proliferation, and remodeling, dysregulation of cholesterol metabolism is associated with multiple age-related pathologies. Here we show that senescent cells accumulate cholesterol in lysosomes to maintain the senescence-associated secretory phenotype (SASP). We find that induction of cellular senescence by diverse triggers enhances cellular cholesterol metabolism. Senescence is associated with the upregulation of the cholesterol exporter ABCA1, which is rerouted to the lysosome, where it moonlights as a cholesterol importer. Lysosomal cholesterol accumulation results in the formation of cholesterol-rich microdomains on the lysosomal limiting membrane enriched with the mammalian target of rapamycin complex 1 (mTORC1) scaffolding complex, thereby sustaining mTORC1 activity to support the SASP. We further show that pharmacological modulation of lysosomal cholesterol partitioning alters senescence-associated inflammation and in vivo senescence during osteoarthritis progression in male mice. Our study reveals a potential unifying theme for the role of cholesterol in the aging process through the regulation of senescence-associated inflammation.
    DOI:  https://doi.org/10.1038/s42255-023-00747-5
  29. Nucleic Acids Res. 2023 Mar 02. pii: gkad116. [Epub ahead of print]
    Proximal binding of dCas9 at a DNA double strand break stimulates homology-directed repair as a local inhibitor of classical non-homologous end joining.
    Yi-Li Feng, Si-Cheng Liu, Ruo-Dan Chen, Xiu-Na Sun, Jing-Jing Xiao, Ji-Feng Xiang, An-Yong Xie.
      In CRISPR/Cas9 genome editing, the tight and persistent target binding of Cas9 provides an opportunity for efficient genetic and epigenetic modification on genome. In particular, technologies based on catalytically dead Cas9 (dCas9) have been developed to enable genomic regulation and live imaging in a site-specific manner. While post-cleavage target residence of CRISPR/Cas9 could alter the pathway choice in repair of Cas9-induced DNA double strand breaks (DSBs), it is possible that dCas9 residing adjacent to a break may also determine the repair pathway for this DSB, providing an opportunity to control genome editing. Here, we found that loading dCas9 onto a DSB-adjacent site stimulated homology-directed repair (HDR) of this DSB by locally blocking recruitment of classical non-homologous end-joining (c-NHEJ) factors and suppressing c-NHEJ in mammalian cells. We further repurposed dCas9 proximal binding to increase HDR-mediated CRISPR genome editing by up to 4-fold while avoiding exacerbation of off-target effects. This dCas9-based local inhibitor provided a novel strategy of c-NHEJ inhibition in CRISPR genome editing in place of small molecule c-NHEJ inhibitors, which are often used to increase HDR-mediated genome editing but undesirably exacerbate off-target effects.
    DOI:  https://doi.org/10.1093/nar/gkad116
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