bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2024–09–22
fourteen papers selected by
Ralitsa Radostinova Madsen, MRC-PPU
  1. Spatial and signaling overlap of growth factor receptor systems at clathrin-coated sites.
  2. Multiplex, single-cell CRISPRa screening for cell type specific regulatory elements.
  3. Understanding the Warburg Effect in Cancer.
  4. Potent combination benefit of the AKT inhibitor capivasertib and the BCL-2 inhibitor venetoclax in diffuse large B cell lymphoma.
  5. Germline mutations in a G protein identify signaling cross-talk in T cells.
  6. Mechanistic target of rapamycin (mTOR) pathway in Sertoli cells regulates age-dependent changes in sperm DNA methylation.
  7. The signaling cascade of induction and maintenance of ES cell diapause.
  8. Small molecules help misplaced proteins hitchhike around cells.
  9. Advances in human pluripotent stem cell reporter systems.
  10. Calibration of FRET-based biosensors using multiplexed biosensor barcoding.
  11. CRISPRi with barcoded expression reporters dissects regulatory networks in human cells.
  12. CRISPR-based dissection of miRNA binding sites using isogenic cell lines is hampered by pervasive noise.
  13. ENCORE: a practical implementation to improve reproducibility and transparency of computational research.
  14. Targeting cancer with small-molecule pan-KRAS degraders.
  1. Mol Biol Cell. 2024 Sep 18. mbcE24050226
    Spatial and signaling overlap of growth factor receptor systems at clathrin-coated sites.
    Marco A Alfonzo-Méndez, Marie-Paule Strub, Justin W Taraska.
      Cellular communication is regulated at the plasma membrane by the interactions of receptor, adhesion, signaling, and endocytic proteins. Yet, the composition and control of these complexes in response to external cues remain unclear. We use high-resolution and high-throughput fluorescence imaging to map the localization of growth factor receptors and related proteins at single clathrin-coated structures in human squamous HSC3 cells. We find distinct protein signatures between control cells and cells stimulated with growth factors. Clathrin sites at the plasma membrane are preloaded with some receptors but not others. Stimulation with epidermal growth factor induces capture and concentration of epidermal growth factor-, fibroblast growth factor-, and low-density lipoprotein-receptors (EGFR, FGFR1, and LDLR). Regulatory proteins including ubiquitin ligase Cbl, the scaffold Grb2, and the mechanoenzyme dynamin2 are also recruited. Disrupting FGFR or EGFR activity with drugs prevents the recruitment of both EGFR and FGFR1. EGF was able to activate FGFR1 phosphorylation. Our data reveals novel co-clustering and activation of receptors and regulatory factors at clathrin-coated sites in response to stimulation by a single growth factor, EGF or FGF. This behavior integrates growth factor signaling and allows for complex responses to extracellular cues and drugs at the plasma membrane of human cells.
    DOI:  https://doi.org/10.1091/mbc.E24-05-0226
  2. Nat Commun. 2024 Sep 18. 15(1): 8209
    Multiplex, single-cell CRISPRa screening for cell type specific regulatory elements.
    Florence M Chardon, Troy A McDiarmid, Nicholas F Page, Riza M Daza, Beth K Martin, Silvia Domcke, Samuel G Regalado, Jean-Benoît Lalanne, Diego Calderon, Xiaoyi Li, Lea M Starita, Stephan J Sanders, Nadav Ahituv, Jay Shendure.
      CRISPR-based gene activation (CRISPRa) is a strategy for upregulating gene expression by targeting promoters or enhancers in a tissue/cell-type specific manner. Here, we describe an experimental framework that combines highly multiplexed perturbations with single-cell RNA sequencing (sc-RNA-seq) to identify cell-type-specific, CRISPRa-responsive cis-regulatory elements and the gene(s) they regulate. Random combinations of many gRNAs are introduced to each of many cells, which are then profiled and partitioned into test and control groups to test for effect(s) of CRISPRa perturbations of both enhancers and promoters on the expression of neighboring genes. Applying this method to a library of 493 gRNAs targeting candidate cis-regulatory elements in both K562 cells and iPSC-derived excitatory neurons, we identify gRNAs capable of specifically upregulating intended target genes and no other neighboring genes within 1 Mb, including gRNAs yielding upregulation of six autism spectrum disorder (ASD) and neurodevelopmental disorder (NDD) risk genes in neurons. A consistent pattern is that the responsiveness of individual enhancers to CRISPRa is restricted by cell type, implying a dependency on either chromatin landscape and/or additional trans-acting factors for successful gene activation. The approach outlined here may facilitate large-scale screens for gRNAs that activate genes in a cell type-specific manner.
    DOI:  https://doi.org/10.1038/s41467-024-52490-4
  3. Cold Spring Harb Perspect Med. 2024 Sep 16. pii: a041532. [Epub ahead of print]
    Understanding the Warburg Effect in Cancer.
    Zhaoqi Li, Muhammad Bin Munim, Daniel A Sharygin, Brooke J Bevis, Matthew G Vander Heiden.
      Rapidly proliferating cells, including cancer cells, adapt metabolism to meet the increased energetic and biosynthetic demands of cell growth and division. Many rapidly proliferating cells exhibit increased glucose consumption and fermentation regardless of oxygen availability, a phenotype termed aerobic glycolysis or the Warburg effect in cancer. Several explanations for why cells engage in aerobic glycolysis and how it supports proliferation have been proposed, but none can fully explain all conditions and data where aerobic glycolysis is observed. Nevertheless, there is convincing evidence that the Warburg effect is important for the proliferation of many cancers, and that inhibiting either glucose uptake or fermentation can impair tumor growth. Here, we discuss what is known about metabolism associated with aerobic glycolysis and the evidence supporting various explanations for why aerobic glycolysis may be important in cancer and other contexts.
    DOI:  https://doi.org/10.1101/cshperspect.a041532
  4. Leukemia. 2024 Sep 16.
    Potent combination benefit of the AKT inhibitor capivasertib and the BCL-2 inhibitor venetoclax in diffuse large B cell lymphoma.
    Brandon S Willis, Kevin Mongeon, Hannah Dry, India L Neveras, Nadezda Bryan, Meghana Pandya, Justine Roderick-Richardson, Wendan Xu, Li Yang, Alan Rosen, Corinne Reimer, Liliana Tuskova, Pavel Klener, Jerome T Mettetal, Georg Lenz, Simon T Barry.
      The therapeutic potential of targeting PI3K/AKT/PTEN signalling in B-cell malignancies remains attractive. Whilst PI3K-α/δ inhibitors demonstrate clinical benefit in certain B-cell lymphomas, PI3K signalling inhibitors have been inadequate in relapsed/refractory diffuse large B-cell lymphoma (DLBCL) in part, due to treatment related toxicities. Clinically, AKT inhibitors exhibit a differentiated tolerability profile offering an alternative approach for treating patients with B-cell malignancies. To explore how AKT inhibition complements other potential therapeutics in the treatment of DLBCL patients, an in vitro combination screen was conducted across a panel of DLCBL cell lines. The AKT inhibitor, capivasertib, in combination with the BCL-2 inhibitor, venetoclax, produced notable therapeutic benefit in preclinical models of DLBCL. Capivasertib and venetoclax rapidly induced caspase and PARP cleavage in GCB-DLBCL PTEN wildtype cell lines and those harbouring PTEN mutations or reduced PTEN protein, driving prolonged tumour growth inhibition in DLBCL cell line and patient derived xenograft lymphoma models. The addition of the rituximab further deepened the durability of capivasertib and venetoclax responses in a RCHOP refractory DLBCL in vivo models. These findings provide preclinical evidence for the rational treatment combination of AKT and BCL-2 inhibitors using capivasertib and venetoclax respectively alongside anti-CD20 antibody supplementation for treatment of patients with DLBCL.
    DOI:  https://doi.org/10.1038/s41375-024-02401-9
  5. Science. 2024 Sep 20. 385(6715): eadd8947
    Germline mutations in a G protein identify signaling cross-talk in T cells.
    Hyoungjun Ham, Huie Jing, Ian T Lamborn, Megan M Kober, Alexey Koval, Yamina A Berchiche, D Eric Anderson, Kirk M Druey, Judith N Mandl, Bertrand Isidor, Carlos R Ferreira, Alexandra F Freeman, Sundar Ganesan, Meliha Karsak, Peter J Mustillo, Juliana Teo, Zarazuela Zolkipli-Cunningham, Nicolas Chatron, François Lecoquierre, Andrew J Oler, Jana Pachlopnik Schmid, Douglas B Kuhns, Xuehua Xu, Fabian Hauck, Waleed Al-Herz, Matias Wagner, Paulien A Terhal, Mari Muurinen, Vincent Barlogis, Phillip Cruz, Jeffrey Danielson, Helen Stewart, Petra Loid, Sebastian Rading, Boris Keren, Rolph Pfundt, Kol A Zarember, Katharina Vill, Lorraine Potocki, Kenneth N Olivier, Gaetan Lesca, Laurence Faivre, Melanie Wong, Anne Puel, Janet Chou, Maud Tusseau, Niki M Moutsopoulos, Helen F Matthews, Cas Simons, Ryan J Taft, Ariane Soldatos, Etienne Masle-Farquhar, Stefania Pittaluga, Robert Brink, Danielle L Fink, Heidi H Kong, Juraj Kabat, Woo Sung Kim, Tatjana Bierhals, Kazuyuki Meguro, Amy P Hsu, Jingwen Gu, Jennifer Stoddard, Benito Banos-Pinero, Maria Slack, Giampaolo Trivellin, Benoît Mazel, Maarja Soomann, Samuel Li, Val J Watts, Constantine A Stratakis, Maria F Rodriguez-Quevedo, Ange-Line Bruel, Marita Lipsanen-Nyman, Paul Saultier, Rashmi Jain, Daphne Lehalle, Daniel Torres, Kathleen E Sullivan, Sébastien Barbarot, Axel Neu, Yannis Duffourd, Morgan Similuk, Kirsty McWalter, Pierre Blanc, Stéphane Bézieau, Tian Jin, Raif S Geha, Jean-Laurent Casanova, Outi M Makitie, Christian Kubisch, Patrick Edery, John Christodoulou, Ronald N Germain, Christopher C Goodnow, Thomas P Sakmar, Daniel D Billadeau, Sébastien Küry, Vladimir L Katanaev, Yu Zhang, Michael J Lenardo, Helen C Su.
      Humans with monogenic inborn errors responsible for extreme disease phenotypes can reveal essential physiological pathways. We investigated germline mutations in GNAI2, which encodes Gαi2, a key component in heterotrimeric G protein signal transduction usually thought to regulate adenylyl cyclase-mediated cyclic adenosine monophosphate (cAMP) production. Patients with activating Gαi2 mutations had clinical presentations that included impaired immunity. Mutant Gαi2 impaired cell migration and augmented responses to T cell receptor (TCR) stimulation. We found that mutant Gαi2 influenced TCR signaling by sequestering the guanosine triphosphatase (GTPase)-activating protein RASA2, thereby promoting RAS activation and increasing downstream extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)-AKT S6 signaling to drive cellular growth and proliferation.
    DOI:  https://doi.org/10.1126/science.add8947
  6. Elife. 2024 Sep 16. pii: RP90992. [Epub ahead of print]13
    Mechanistic target of rapamycin (mTOR) pathway in Sertoli cells regulates age-dependent changes in sperm DNA methylation.
    Saira Amir, Olatunbosun Arowolo, Ekaterina Mironova, Joseph McGaunn, Oladele Oluwayiose, Oleg Sergeyev, J Richard Pilsner, Alexander Suvorov.
      Over the past several decades, a trend toward delayed childbirth has led to increases in parental age at the time of conception. Sperm epigenome undergoes age-dependent changes increasing risks of adverse conditions in offspring conceived by fathers of advanced age. The mechanism(s) linking paternal age with epigenetic changes in sperm remain unknown. The sperm epigenome is shaped in a compartment protected by the blood-testes barrier (BTB) known to deteriorate with age. Permeability of the BTB is regulated by the balance of two mTOR complexes in Sertoli cells where mTOR complex 1 (mTORC1) promotes the opening of the BTB and mTOR complex 2 (mTORC2) promotes its integrity. We hypothesized that this balance is also responsible for age-dependent changes in the sperm epigenome. To test this hypothesis, we analyzed reproductive outcomes, including sperm DNA methylation in transgenic mice with Sertoli cell-specific suppression of mTORC1 (Rptor KO) or mTORC2 (Rictor KO). mTORC2 suppression accelerated aging of the sperm DNA methylome and resulted in a reproductive phenotype concordant with older age, including decreased testes weight and sperm counts, and increased percent of morphologically abnormal spermatozoa and mitochondrial DNA copy number. Suppression of mTORC1 resulted in the shift of DNA methylome in sperm opposite to the shift associated with physiological aging - sperm DNA methylome rejuvenation and mild changes in sperm parameters. These results demonstrate for the first time that the balance of mTOR complexes in Sertoli cells regulates the rate of sperm epigenetic aging. Thus, mTOR pathway in Sertoli cells may be used as a novel target of therapeutic interventions to rejuvenate the sperm epigenome in advanced-age fathers.
    Keywords:  DNA methylation; aging; developmental biology; epigenetic; mTOR; mouse; rejuvenation; sperm
    DOI:  https://doi.org/10.7554/eLife.90992
  7. Res Sq. 2024 Sep 03. pii: rs.3.rs-4946357. [Epub ahead of print]
    The signaling cascade of induction and maintenance of ES cell diapause.
    Alexander Tarakhovsky, Tuo Zhang, Ryan Marina, Sarah Veugelen, Pal Mander, Rabinder Prinjha, Anne Schaefer, Karen Adelman.
      Nutrient deficiency during pregnancy in numerous animal species can induce the state of embryonic diapause. Diapause is characterized by changes in protein and gene expression that minimize the organism's reliance on external energy sources and ensure survival. Remarkably, the systematic changes associated with diapause appear to spare the gene expression program that supports embryonic cells' maintenance in the pluripotent state. The phenomenon of the differentiation "freeze" during diapause can be reproduced in vitro . Mimicking nutrient deficiency by pharmacological inhibition of mTOR induces the diapause-like state in ES cells without affecting ES cell pluripotency. We discovered a connection between mTOR signaling and the chromatin-bound bromodomain and extra-terminal (BET) transcriptional regulator BRD4, showing a key role of BET-protein in the induction of diapause-like state in ES cells. mTOR inhibition rapidly and negatively impacts BRD4 binding to chromatin, which is associated with changes in gene expression that can contribute to diapause. Conversely, pharmacological inhibition of BET-protein circumvents the diapause dependence on mTOR inhibition and causes the diapause-like state. BET-repressed diapause-like ES cells retain the undifferentiated pluripotent state, which is associated with upregulation of a functionally linked group of genes encoding negative regulators of MAP kinase (MAPK) signaling and inactivation of MAP kinase. The transcriptional switch-off of MAP kinase following chronic BET inhibition imitates the transcriptional de-repression of MAP kinase negative regulators in response to mTOR inhibition. Mechanistically, suppression of mTOR or BET-protein leads to a profound decline in Capicua transcriptional repressor (CIC) at promoters of key negative regulators of MAP kinase. The discovered mTOR-BRD4 axis in the induction of diapause and the rapid transcriptional shut-off of differentiation program is likely to play a major role in the maintenance of embryonic diapause in vivo , as well as in controlling of the undifferentiated state of various types of stem cells during diapause-like metabolic dormancy.
    DOI:  https://doi.org/10.21203/rs.3.rs-4946357/v1
  8. Nature. 2024 Sep 18.
    Small molecules help misplaced proteins hitchhike around cells.
    Robert Yvon, Christina M Woo.
      
    Keywords:  Chemical biology
    DOI:  https://doi.org/10.1038/d41586-024-02802-x
  9. iScience. 2024 Sep 20. 27(9): 110856
    Advances in human pluripotent stem cell reporter systems.
    Lesly Puspita, Virginia Blessy Juwono, Jae-Won Shim.
      The capability of human pluripotent stem cells (hPSCs) to self-renew and differentiate into any cell type has greatly contributed to the advancement of biomedicine. Reporter lines derived from hPSCs have played a crucial role in elucidating the mechanisms underlying human development and diseases by acting as an alternative reporter system that cannot be used in living humans. To bring hPSCs closer to clinical application in transplantation, scientists have generated reporter lines for isolating the desired cell populations, as well as improving graft quality and treatment outcomes. This review presents an overview of the applications of hPSC reporter lines and the important variables in designing a reporter system, including options for gene delivery and editing tools, design of reporter constructs, and selection of reporter genes. It also provides insights into the prospects of hPSC reporter lines and the challenges that must be overcome to maximize the potential of hPSC reporter lines.
    Keywords:  Methodology in biological sciences; Molecular biology experimental approach; Stem cells research
    DOI:  https://doi.org/10.1016/j.isci.2024.110856
  10. bioRxiv. 2024 Sep 08. pii: 2024.09.04.610346. [Epub ahead of print]
    Calibration of FRET-based biosensors using multiplexed biosensor barcoding.
    Jhen-Wei Wu, Jr-Ming Yang, Chao-Cheng Chen, Gabriel Au, Suyang Wang, Gia-Wei Chern, Chuan-Hsiang Huang.
      Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) is widely used in the design of genetically encoded fluorescent biosensors, which are powerful tools for monitoring the dynamics of biochemical activities in live cells. FRET ratio, defined as the ratio between acceptor and donor signals, is often used as a proxy for the actual FRET efficiency, which must be corrected for signal crosstalk using donor-only and acceptor-only samples. However, the FRET ratio is highly sensitive to imaging conditions, making direct comparisons across different experiments and over time challenging. Inspired by a method for multiplexed biosensor imaging using barcoded cells, we reasoned that calibration standards with fixed FRET efficiency can be introduced into a subset of cells for normalization of biosensor signals. Our theoretical analysis indicated that the FRET ratio of high-FRET species relative to non-FRET species slightly decreases at high excitation intensity, suggesting the need for calibration using both high and low FRET standards. To test these predictions, we created FRET donor-acceptor pairs locked in "FRET-ON" and "FRET-OFF" conformations and introduced them into a subset of barcoded cells. Our results confirmed the theoretical predictions and showed that the calibrated FRET ratio is independent of imaging settings. We also provided a strategy for calculating the FRET efficiency. Together, our study presents a simple strategy for calibrated and highly multiplexed imaging of FRET biosensors, facilitating reliable comparisons across experiments and supporting long-term imaging applications.
    DOI:  https://doi.org/10.1101/2024.09.04.610346
  11. bioRxiv. 2024 Sep 06. pii: 2024.09.06.611573. [Epub ahead of print]
    CRISPRi with barcoded expression reporters dissects regulatory networks in human cells.
    Jinyoung Kim, Ryan Y Muller, Eliana R Bondra, Nicholas T Ingolia.
      Genome-wide CRISPR screens have emerged as powerful tools for uncovering the genetic underpinnings of diverse biological processes. Incisive screens often depend on directly measuring molecular phenotypes, such as regulated gene expression changes, provoked by CRISPR-mediated genetic perturbations. Here, we provide quantitative measurements of transcriptional responses in human cells across genome-scale perturbation libraries by coupling CRISPR interference (CRISPRi) with barcoded expression reporter sequencing (CiBER-seq). To enable CiBER-seq in mammalian cells, we optimize the integration of highly complex, barcoded sgRNA libraries into a defined genomic context. CiBER-seq profiling of a nuclear factor kappa B (NF-κB) reporter delineates the canonical signaling cascade linking the transmembrane TNF-alpha receptor to inflammatory gene activation and highlights cell-type-specific factors in this response. Importantly, CiBER-seq relies solely on bulk RNA sequencing to capture the regulatory circuit driving this rapid transcriptional response. Our work demonstrates the accuracy of CiBER-seq and its potential for dissecting genetic networks in mammalian cells with superior time resolution.
    DOI:  https://doi.org/10.1101/2024.09.06.611573
  12. bioRxiv. 2024 Sep 03. pii: 2024.09.03.611048. [Epub ahead of print]
    CRISPR-based dissection of miRNA binding sites using isogenic cell lines is hampered by pervasive noise.
    Mahendra K Prajapat, Joana A Vidigal.
      Non-coding regulatory sequences play essential roles in adjusting gene output to cellular needs and are thus critical to animal development and health. Numerous such sequences have been identified in mammalian genomes ranging from transcription factors binding motifs to recognition sites for RNA-binding proteins and non-coding RNAs. The advent of CRISPR has raised the possibility of assigning functionality to individual endogenous regulatory sites by facilitating the generation of isogenic cell lines that differ by a defined set of genetic modifications. Here we investigate the usefulness of this approach to assign function to individual miRNA binding sites. We find that the process of generating isogenic pairs of mammalian cell lines with CRISPR-mediated mutations introduces extensive molecular and phenotypic variability between biological replicates making any attempt of assigning function to the binding site essentially impossible. Our work highlights an important consideration when employing CRISPR editing to characterize non-coding regulatory sequences in cell lines and calls for the development and adoption of alternative strategies to address this question in the future.
    DOI:  https://doi.org/10.1101/2024.09.03.611048
  13. Nat Commun. 2024 Sep 16. 15(1): 8117
    ENCORE: a practical implementation to improve reproducibility and transparency of computational research.
    Antoine H C van Kampen, Utkarsh Mahamune, Aldo Jongejan, Barbera D C van Schaik, Daria Balashova, Danial Lashgari, Mia Pras-Raves, Eric J M Wever, Adrie D Dane, Rodrigo García-Valiente, Perry D Moerland.
      Reproducibility of computational research is often challenging despite established guidelines and best practices. Translating these guidelines into practical applications remains difficult. Here, we present ENCORE, an approach to enhance transparency and reproducibility by guiding researchers in how to structure and document a computational project. ENCORE builds on previous efforts in computational reproducibility and integrates all project components into a standardized file system structure. It utilizes pre-defined files as documentation templates, leverages GitHub for software versioning, and includes an HTML-based navigator. ENCORE is designed to be agnostic to the type of computational project, data, programming language, and ICT infrastructure, and does not rely on specific software tools. We also share our group's experience using ENCORE, highlighting that the most significant challenge to the routine adoption of approaches like ours is the lack of incentives to motivate researchers to dedicate sufficient time and effort to ensure reproducibility.
    DOI:  https://doi.org/10.1038/s41467-024-52446-8
  14. Science. 2024 Sep 20. 385(6715): 1338-1347
    Targeting cancer with small-molecule pan-KRAS degraders.
    Johannes Popow, William Farnaby, Andreas Gollner, Christiane Kofink, Gerhard Fischer, Melanie Wurm, David Zollman, Andre Wijaya, Nikolai Mischerikow, Carina Hasenoehrl, Polina Prokofeva, Heribert Arnhof, Silvia Arce-Solano, Sammy Bell, Georg Boeck, Emelyne Diers, Aileen B Frost, Jake Goodwin-Tindall, Jale Karolyi-Oezguer, Shakil Khan, Theresa Klawatsch, Manfred Koegl, Roland Kousek, Barbara Kratochvil, Katrin Kropatsch, Arnel A Lauber, Ross McLennan, Sabine Olt, Daniel Peter, Oliver Petermann, Vanessa Roessler, Peggy Stolt-Bergner, Patrick Strack, Eva Strauss, Nicole Trainor, Vesna Vetma, Claire Whitworth, Siying Zhong, Jens Quant, Harald Weinstabl, Bernhard Kuster, Peter Ettmayer, Alessio Ciulli.
      Mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) protein are highly prevalent in cancer. However, small-molecule concepts that address oncogenic KRAS alleles remain elusive beyond replacing glycine at position 12 with cysteine (G12C), which is clinically drugged through covalent inhibitors. Guided by biophysical and structural studies of ternary complexes, we designed a heterobifunctional small molecule that potently degrades 13 out of 17 of the most prevalent oncogenic KRAS alleles. Compared with inhibition, KRAS degradation results in more profound and sustained pathway modulation across a broad range of KRAS mutant cell lines, killing cancer cells while sparing models without genetic KRAS aberrations. Pharmacological degradation of oncogenic KRAS was tolerated and led to tumor regression in vivo. Together, these findings unveil a new path toward addressing KRAS-driven cancers with small-molecule degraders.
    DOI:  https://doi.org/10.1126/science.adm8684
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