bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2023–04–16
nineteen papers selected by
Ralitsa Radostinova Madsen, MRC-PPU
  1. AKT activity orchestrates marginal zone B cell development in mice and humans.
  2. The scverse project provides a computational ecosystem for single-cell omics data analysis.
  3. Uncovering biology by single-cell proteomics.
  4. Phospho-seq: Integrated, multi-modal profiling of intracellular protein dynamics in single cells.
  5. Single-cell measurement of dynamic protein secretion and transcriptome.
  6. N-acetylcysteine overcomes NF1 loss-driven resistance to PI3Kα inhibition in breast cancer.
  7. Genome-wide CRISPR screens identify ILF3 as a mediator of mTORC1-dependent amino acid sensing.
  8. Redundant roles of AKT1 and AKT2 in fertility, estrous cyclicity and endometrial gland development.
  9. SUMOylation and the oncogenic E17K mutation affect AKT1 subcellular distribution and impact on Nanog-binding dynamics to chromatin in embryonic stem cells.
  10. Applying the Cytocentric Principles to Regenerative Medicine for Reproducibility.
  11. Local generation and efficient evaluation of numerous drug combinations in a single sample.
  12. SEQUIN is an R/Shiny framework for rapid and reproducible analysis of RNA-seq data.
  13. Mechanosensitive mTORC1 signaling maintains lymphatic valves.
  14. FDA approves PI3K inhibitor for a rare immune disorder.
  15. Comparison of transformations for single-cell RNA-seq data.
  16. High-efficiency and multilocus targeted integration in CHO cells using CRISPR-mediated donor nicking and DNA repair inhibitors.
  17. The origins and functional effects of postzygotic mutations throughout the human life span.
  18. Cancer: Untangling complexity together.
  19. Lipid droplets are intracellular mechanical stressors that impair hepatocyte function.
  1. Cell Rep. 2023 Apr 14. pii: S2211-1247(23)00389-3. [Epub ahead of print]42(4): 112378
    AKT activity orchestrates marginal zone B cell development in mice and humans.
    Eva-Maria Cox, Mohamed El-Behi, Stefanie Ries, Johannes F Vogt, Vivien Kohlhaas, Thomas Michna, Benoît Manfroi, Mona Al-Maarri, Florian Wanke, Boaz Tirosh, Corinne Pondarre, Harry Lezeau, Nir Yogev, Romy Mittenzwei, Marc Descatoire, Sandra Weller, Jean-Claude Weill, Claude-Agnès Reynaud, Pierre Boudinot, Luc Jouneau, Stefan Tenzer, Ute Distler, Anne Rensing-Ehl, Christoph König, Julian Staniek, Marta Rizzi, Aude Magérus, Frederic Rieux-Laucat, F Thomas Wunderlich, Nadine Hövelmeyer, Simon Fillatreau.
      The signals controlling marginal zone (MZ) and follicular (FO) B cell development remain incompletely understood. Here, we show that AKT orchestrates MZ B cell formation in mice and humans. Genetic models that increase AKT signaling in B cells or abolish its impact on FoxO transcription factors highlight the AKT-FoxO axis as an on-off switch for MZ B cell formation in mice. In humans, splenic immunoglobulin (Ig) D+CD27+ B cells, proposed as an MZ B cell equivalent, display higher AKT signaling than naive IgD+CD27- and memory IgD-CD27+ B cells and develop in an AKT-dependent manner from their precursors in vitro, underlining the conservation of this developmental pathway. Consistently, CD148 is identified as a receptor indicative of the level of AKT signaling in B cells, expressed at a higher level in MZ B cells than FO B cells in mice as well as humans.
    Keywords:  AKT; ALPS; B cells; CD148; CP: Developmental biology; CP: Immunology; FoxO1; NOTCH2; marginal zone B cells
    DOI:  https://doi.org/10.1016/j.celrep.2023.112378
  2. Nat Biotechnol. 2023 Apr 10.
    The scverse project provides a computational ecosystem for single-cell omics data analysis.
    Scverse Community
      
    DOI:  https://doi.org/10.1038/s41587-023-01733-8
  3. Commun Biol. 2023 04 08. 6(1): 381
    Uncovering biology by single-cell proteomics.
    M Shahid Mansuri, Kenneth Williams, Angus C Nairn.
      Recent technological advances have opened the door to single-cell proteomics that can answer key biological questions regarding how protein expression, post-translational modifications, and protein interactions dictate cell state in health and disease.
    DOI:  https://doi.org/10.1038/s42003-023-04635-2
  4. bioRxiv. 2023 Mar 28. pii: 2023.03.27.534442. [Epub ahead of print]
    Phospho-seq: Integrated, multi-modal profiling of intracellular protein dynamics in single cells.
    John D Blair, Austin Hartman, Fides Zenk, Carol Dalgarno, Barbara Treutlein, Rahul Satija.
      Cell signaling plays a critical role in regulating cellular behavior and fate. While multimodal single-cell sequencing technologies are rapidly advancing, scalable and flexible profiling of cell signaling states alongside other molecular modalities remains challenging. Here we present Phospho-seq, an integrated approach that aims to quantify phosphorylated intracellular and intranuclear proteins, and to connect their activity with cis-regulatory elements and transcriptional targets. We utilize a simplified benchtop antibody conjugation method to create large custom antibody panels for simultaneous protein and scATAC-seq profiling on whole cells, and integrate this information with scRNA-seq datasets via bridge integration. We apply our workflow to cell lines, induced pluripotent stem cells, and 3-month-old brain organoids to demonstrate its broad applicability. We demonstrate that Phospho-seq can define cellular states and trajectories, reconstruct gene regulatory relationships, and characterize the causes and consequences of heterogeneous cell signaling in neurodevelopment.
    DOI:  https://doi.org/10.1101/2023.03.27.534442
  5. Nat Methods. 2023 Apr 11.
    Single-cell measurement of dynamic protein secretion and transcriptome.
      
    DOI:  https://doi.org/10.1038/s41592-023-01842-x
  6. Cell Rep Med. 2023 Apr 04. pii: S2666-3791(23)00112-X. [Epub ahead of print] 101002
    N-acetylcysteine overcomes NF1 loss-driven resistance to PI3Kα inhibition in breast cancer.
    Priska Auf der Maur, Marcel P Trefny, Zora Baumann, Milica Vulin, Ana Luisa Correia, Maren Diepenbruck, Nicolas Kramer, Katrin Volkmann, Bogdan-Tiberius Preca, Pedro Ramos, Cedric Leroy, Tobias Eichlisberger, Katarzyna Buczak, Federica Zilli, Ryoko Okamoto, Roland Rad, Michael Rugaard Jensen, Christine Fritsch, Alfred Zippelius, Michael B Stadler, Mohamed Bentires-Alj.
      A genome-wide PiggyBac transposon-mediated screen and a resistance screen in a PIK3CAH1047R-mutated murine tumor model reveal NF1 loss in mammary tumors resistant to the phosphatidylinositol 3-kinase α (PI3Kα)-selective inhibitor alpelisib. Depletion of NF1 in PIK3CAH1047R breast cancer cell lines and a patient-derived organoid model shows that NF1 loss reduces sensitivity to PI3Kα inhibition and correlates with enhanced glycolysis and lower levels of reactive oxygen species (ROS). Unexpectedly, the antioxidant N-acetylcysteine (NAC) sensitizes NF1 knockout cells to PI3Kα inhibition and reverts their glycolytic phenotype. Global phospho-proteomics indicates that combination with NAC enhances the inhibitory effect of alpelisib on mTOR signaling. In public datasets of human breast cancer, we find that NF1 is frequently mutated and that such mutations are enriched in metastases, an indication for which use of PI3Kα inhibitors has been approved. Our results raise the attractive possibility of combining PI3Kα inhibition with NAC supplementation, especially in patients with drug-resistant metastases associated with NF1 loss.
    Keywords:  PI3K alpha inhibition; breast cancer; combination therapy; resistance; transposon screen
    DOI:  https://doi.org/10.1016/j.xcrm.2023.101002
  7. Nat Cell Biol. 2023 Apr 10.
    Genome-wide CRISPR screens identify ILF3 as a mediator of mTORC1-dependent amino acid sensing.
    Guokai Yan, Jinxin Yang, Wen Li, Ao Guo, Jialiang Guan, Ying Liu.
      The mechanistic target of rapamycin complex 1 (mTORC1) is an essential hub that integrates nutrient signals and coordinates metabolism to control cell growth. Amino acid signals are detected by sensor proteins and relayed to the GATOR2 and GATOR1 complexes to control mTORC1 activity. Here we perform genome-wide CRISPR/Cas9 screens, coupled with an assay for mTORC1 activity based on fluorescence-activated cell sorting analysis of pS6, to identify potential regulators of mTORC1-dependent amino acid sensing. We then focus on interleukin enhancer binding factor 3 (ILF3), one of the candidate genes from the screen. ILF3 tethers the GATOR complexes to lysosomes to control mTORC1. Adding a lysosome-targeting sequence to the GATOR2 component WDR24 bypasses the requirement for ILF3 to modulate amino-acid-dependent mTORC1 signalling. ILF3 plays an evolutionarily conserved role in human and mouse cells, and in worms to regulate the mTORC1 pathway, control autophagy activity and modulate the ageing process.
    DOI:  https://doi.org/10.1038/s41556-023-01123-x
  8. Reproduction. 2023 Apr 01. pii: REP-22-0353. [Epub ahead of print]
    Redundant roles of AKT1 and AKT2 in fertility, estrous cyclicity and endometrial gland development.
    Pascal Adam, Laurence Tardif, François Fabi, Dadou Likonza Lokengo, Léa-Isabelle Renaud, Sophie Parent, Monique Cadrin, Derek Boerboom, Eric Asselin.
      Apoptosis and cell survival regulation are crucial processes during the estrous cycle to prepare a receptive uterus during implantation for successful recognition of pregnancy. PI3K/AKT signaling has a crucial role during gestation and AKT isoforms (1, 2 or 3) are regulated differently in the endometrium during the estrous cycle and embryo implantation. However, the specific roles of these isoforms are still unclear. We have previously shown that AKT isoforms expression during the rat estrous cycle and gestation are differently regulated. The present study aimed to establish the specific role of AKT isoforms in the mouse uterus. The hypothesis is that a dysregulation of AKT isoforms expression could cause fertility-related issues in an isoform-specific manner. With four different mouse models and in-house crossbreeding, all isoforms KO combinations (single, double and triple) were obtained in PGR expressing tissues. The results demonstrated that in absence of one or more AKT isoforms, female fertility was decreased. Mainly, we have observed smaller litter size, specifically in Akt1-2 KO mice. Additionally, we have found Akt1-2-3 KO mice to be fully infertile. Estrous cyclicity was also disrupted in Akt1-2 KO mice with longer diestrus stage. Moreover, the number of endometrial glands was decreased throughout the estrous cycle suggesting an important role in gland development for AKT1 and AKT2. Our results suggest specific roles between each isoform, but also a partially redundant function of AKT1 and AKT2 in litter size, estrous cyclicity and endometrial gland development. This highlights the importance of AKT in the physiological regulation of mouse fertility.
    DOI:  https://doi.org/10.1530/REP-22-0353
  9. J Struct Biol. 2023 Apr 12. pii: S1047-8477(23)00024-2. [Epub ahead of print] 107961
    SUMOylation and the oncogenic E17K mutation affect AKT1 subcellular distribution and impact on Nanog-binding dynamics to chromatin in embryonic stem cells.
    Marcos Gabriel Francia, Camila Oses, Sabrina Lorena Roberti, Mora Reneé Garcia, Lucas Helio Cozza, Maria Candelaria Diaz, Valeria Levi, Alejandra Sonia Guberman.
      AKT/PKB is a kinase involved in the regulation of a plethora of cell processes. Particularly, in embryonic stem cells (ESCs), AKT is crucial for the maintenance of pluripotency. Although the activation of this kinase relies on its recruitment to the cellular membrane and subsequent phosphorylation, multiple other post-translational modifications (PTMs), including SUMOylation, fine-tune its activity and target specificity. Since this PTM can also modify the localization and availability of different proteins, in this work we explored if SUMOylation impacts on the subcellular compartmentalization and distribution of AKT1 in ESCs. We found that this PTM does not affect AKT1 membrane recruitment, but it modifies the AKT1 nucleus/cytoplasm distribution, increasing its nuclear presence. Additionally, within this compartment, we found that AKT1 SUMOylation also impacts on the chromatin-binding dynamics of NANOG, a central pluripotency transcription factor. Remarkably, the oncogenic E17K AKT1 mutant produces major changes in all these parameters increasing the binding of NANOG to its targets, also in a SUMOylation dependent manner. These findings demonstrate that SUMOylation modulates AKT1 subcellular distribution, thus adding an extra layer of regulation of its function, possibly by affecting the specificity and interaction with its downstream targets.
    Keywords:  AKT SUMOylation; AKT distribution; E17K mutant; FCS; Myr AKT; Nanog
    DOI:  https://doi.org/10.1016/j.jsb.2023.107961
  10. Curr Stem Cell Rep. 2022 ;8(4):
    Applying the Cytocentric Principles to Regenerative Medicine for Reproducibility.
    Alicia D Henn, Kunal Mitra, Joshua Hunsberger, Xiuzhi Susan Sun, Mark Nardone, Ramon Montero, Sita Somara, Gary Green, Alan Blanchard, Yu Shrike Zhang, Carl G Simon, Randy Yerden.
       Purpose of Review: Cell and tissue products do not just reflect their present conditions; they are the culmination of all they have encountered over time. Currently, routine cell culture practices subject cell and tissue products to highly variable and non-physiologic conditions. This article defines five cytocentric principles that place the conditions for cells at the core of what we do for better reproducibility in Regenerative Medicine.
    Recent Findings: There is a rising awareness of the cell environment as a neglected, but critical variable. Recent publications have called for controlling culture conditions for better, more reproducible cell products.
    Summary: Every industry has basic quality principles for reproducibility. Cytocentric principles focus on the fundamental needs of cells: protection from contamination, physiologic simulation, and full-time conditions for cultures that are optimal, individualized, and dynamic. Here, we outline the physiologic needs, the technologies, the education, and the regulatory support for the cytocentric principles in regenerative medicine.
    Keywords:  Cell culture; Cytocentric; Organoids; Regenerative medicine; Stem cells; Tissue engineering
    DOI:  https://doi.org/10.1007/s40778-022-00219-8
  11. Elife. 2023 Apr 11. pii: e85439. [Epub ahead of print]12
    Local generation and efficient evaluation of numerous drug combinations in a single sample.
    Vlad Elgart, Joseph Loscalzo.
      We develop a method that allows one to test a large number of drug combinations in a single cell culture sample. We rely on the randomness of drug uptake in individual cells as a tool to create and encode drug treatment regimens. A single sample containing thousands of cells is treated with a combination of fluorescently barcoded drugs. We create independent transient drug gradients across the cell culture sample to produce heterogeneous local drug combinations. After the incubation period, the ensuing phenotype and corresponding drug barcodes for each cell are recorded. We use these data for statistical prediction of the treatment response to the drugs in a macroscopic population of cells. To further application of this technology, we developed a fluorescent barcodingmethod that does not require any chemical drug(s) modifications. We also developed segmentation-free image analysis capable of handling large optical fields containing thousands of cells in the sample, even in confluent growth condition. The technology necessary to execute our method is readily available in most biological laboratories, does not require robotic or microfluidic devices, and dramatically reduces resource needs and resulting costs of the traditional high-throughput studies.
    Keywords:  computational biology; human; medicine; systems biology
    DOI:  https://doi.org/10.7554/eLife.85439
  12. Cell Rep Methods. 2023 Mar 27. 3(3): 100420
    SEQUIN is an R/Shiny framework for rapid and reproducible analysis of RNA-seq data.
    Claire Weber, Marissa B Hirst, Ben Ernest, Nicholas J Schaub, Kelli M Wilson, Ke Wang, Hannah M Baskir, Pei-Hsuan Chu, Carlos A Tristan, Ilyas Singeç.
      SEQUIN is a web-based application (app) that allows fast and intuitive analysis of RNA sequencing data derived for model organisms, tissues, and single cells. Integrated app functions enable uploading datasets, quality control, gene set enrichment, data visualization, and differential gene expression analysis. We also developed the iPSC Profiler, a practical gene module scoring tool that helps measure and compare pluripotent and differentiated cell types. Benchmarking to other commercial and non-commercial products underscored several advantages of SEQUIN. Freely available to the public, SEQUIN empowers scientists using interdisciplinary methods to investigate and present transcriptome data firsthand with state-of-the-art statistical methods. Hence, SEQUIN helps democratize and increase the throughput of interrogating biological questions using next-generation sequencing data with single-cell resolution.
    Keywords:  R/Shiny app; RNA sequencing; UMAP; data visualization; dimensionality reduction; gene expression; iPSC profiler; single-cell analysis; t-SNE; transcriptome analysis
    DOI:  https://doi.org/10.1016/j.crmeth.2023.100420
  13. J Cell Biol. 2023 Jun 05. pii: e202207049. [Epub ahead of print]222(6):
    Mechanosensitive mTORC1 signaling maintains lymphatic valves.
    Cansaran Saygili Demir, Amélie Sabine, Muyun Gong, Olivier Dormond, Tatiana V Petrova.
      Homeostatic maintenance and repair of lymphatic vessels are essential for health. We investigated the dynamics and the molecular mechanisms of lymphatic endothelial cell (LEC) renewal in adult mesenteric quiescent lymphatic vasculature using label-retention, lineage tracing, and cell ablation strategies. Unlike during development, adult LEC turnover and proliferation was confined to the valve regions of collecting vessels, with valve cells displaying the shortest lifespan. Proliferating valve sinus LECs were the main source for maintenance and repair of lymphatic valves. We identified mechanistic target of rapamycin complex 1 (mTORC1) as a mechanoresponsive pathway activated by fluid shear stress in LECs. Depending on the shear stress level, mTORC1 activity drives division of valve cells or dictates their mechanic resilience through increased protein synthesis. Overactivation of lymphatic mTORC1 in vivo promoted supernumerary valve formation. Our work provides insights into the molecular mechanisms of maintenance of healthy lymphatic vascular system.
    DOI:  https://doi.org/10.1083/jcb.202207049
  14. Nat Rev Drug Discov. 2023 Apr 11.
    FDA approves PI3K inhibitor for a rare immune disorder.
    Asher Mullard.
      
    DOI:  https://doi.org/10.1038/d41573-023-00061-5
  15. Nat Methods. 2023 Apr 10.
    Comparison of transformations for single-cell RNA-seq data.
    Constantin Ahlmann-Eltze, Wolfgang Huber.
      The count table, a numeric matrix of genes × cells, is the basic input data structure in the analysis of single-cell RNA-sequencing data. A common preprocessing step is to adjust the counts for variable sampling efficiency and to transform them so that the variance is similar across the dynamic range. These steps are intended to make subsequent application of generic statistical methods more palatable. Here, we describe four transformation approaches based on the delta method, model residuals, inferred latent expression state and factor analysis. We compare their strengths and weaknesses and find that the latter three have appealing theoretical properties; however, in benchmarks using simulated and real-world data, it turns out that a rather simple approach, namely, the logarithm with a pseudo-count followed by principal-component analysis, performs as well or better than the more sophisticated alternatives. This result highlights limitations of current theoretical analysis as assessed by bottom-line performance benchmarks.
    DOI:  https://doi.org/10.1038/s41592-023-01814-1
  16. Biotechnol Bioeng. 2023 Apr 11.
    High-efficiency and multilocus targeted integration in CHO cells using CRISPR-mediated donor nicking and DNA repair inhibitors.
    Nathaniel K Hamaker, Kelvin H Lee.
      Efforts to leverage clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) for targeted genomic modifications in mammalian cells are limited by low efficiencies and heterogeneous outcomes. To aid method optimization, we developed an all-in-one reporter system, including a novel superfolder orange fluorescent protein (sfOrange), to simultaneously quantify gene disruption, site-specific integration (SSI), and random integration (RI). SSI strategies that utilize different donor plasmid formats and Cas9 nuclease variants were evaluated for targeting accuracy and efficiency in Chinese hamster ovary cells. Double-cut and double-nick donor formats significantly improved targeting accuracy by 2.3-8.3-fold and 19-22-fold, respectively, compared to standard circular donors. Notably, Cas9-mediated donor linearization was associated with increased RI events, whereas donor nicking minimized RI without sacrificing SSI efficiency and avoided low-fidelity outcomes. A screen of 10 molecules that modulate the major mammalian DNA repair pathways identified two inhibitors that further enhance targeting accuracy and efficiency to achieve SSI in 25% of transfected cells without selection. The optimized methods integrated transgene expression cassettes with 96% efficiency at a single locus and with 53%-55% efficiency at two loci simultaneously in selected clones. The CRISPR-based tools and methods developed here could inform the use of CRISPR/Cas9 in mammalian cell lines, accelerate mammalian cell line engineering, and support advanced recombinant protein production applications.
    Keywords:  Chinese hamster ovary cell; DNA repair; cell line development; recombinase-mediated cassette exchange; sfOrange; site-specific integration
    DOI:  https://doi.org/10.1002/bit.28393
  17. Science. 2023 Apr 14. 380(6641): eabn7113
    The origins and functional effects of postzygotic mutations throughout the human life span.
    Nicole B Rockweiler, Avinash Ramu, Liina Nagirnaja, Wing H Wong, Michiel J Noordam, Casey W Drubin, Ni Huang, Brian Miller, Ellen Z Todres, Katinka A Vigh-Conrad, Antonino Zito, Kerrin S Small, Kristin G Ardlie, Barak A Cohen, Donald F Conrad.
      Postzygotic mutations (PZMs) begin to accrue in the human genome immediately after fertilization, but how and when PZMs affect development and lifetime health remain unclear. To study the origins and functional consequences of PZMs, we generated a multitissue atlas of PZMs spanning 54 tissue and cell types from 948 donors. Nearly half the variation in mutation burden among tissue samples can be explained by measured technical and biological effects, and 9% can be attributed to donor-specific effects. Through phylogenetic reconstruction of PZMs, we found that their type and predicted functional impact vary during prenatal development, across tissues, and through the germ cell life cycle. Thus, methods for interpreting effects across the body and the life span are needed to fully understand the consequences of genetic variants.
    DOI:  https://doi.org/10.1126/science.abn7113
  18. Cell. 2023 Apr 13. pii: S0092-8674(23)00284-2. [Epub ahead of print]186(8): 1513-1514
    Cancer: Untangling complexity together.
    Cell editorial team
      
    DOI:  https://doi.org/10.1016/j.cell.2023.03.021
  19. Proc Natl Acad Sci U S A. 2023 Apr 18. 120(16): e2216811120
    Lipid droplets are intracellular mechanical stressors that impair hepatocyte function.
    Abigail E Loneker, Farid Alisafaei, Aayush Kant, David Li, Paul A Janmey, Vivek B Shenoy, Rebecca G Wells.
      Matrix stiffening and external mechanical stress have been linked to disease and cancer development in multiple tissues, including the liver, where cirrhosis (which increases stiffness markedly) is the major risk factor for hepatocellular carcinoma. Patients with nonalcoholic fatty liver disease and lipid droplet-filled hepatocytes, however, can develop cancer in noncirrhotic, relatively soft tissue. Here, by treating primary human hepatocytes with the monounsaturated fatty acid oleate, we show that lipid droplets are intracellular mechanical stressors with similar effects to tissue stiffening, including nuclear deformation, chromatin condensation, and impaired hepatocyte function. Mathematical modeling of lipid droplets as inclusions that have only mechanical interactions with other cellular components generated results consistent with our experiments. These data show that lipid droplets are intracellular sources of mechanical stress and suggest that nuclear membrane tension integrates cell responses to combined internal and external stresses.
    Keywords:  HNF4α; chromatin condensation; cytoskeleton; mechanobiology; nuclear deformation
    DOI:  https://doi.org/10.1073/pnas.2216811120
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