bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2023–12–31
twelve papers selected by
Ralitsa Radostinova Madsen, MRC-PPU
  1. Actin-binding protein Profilin1 is an important determinant of cellular phosphoinositide control.
  2. Modulating the PI3K Signalling Pathway in Activated PI3K Delta Syndrome: a Clinical Perspective.
  3. Signaling plasticity in the integrated stress response.
  4. Rescuing the cytolytic function of APDS1 patient T cells via TALEN-mediated PIK3CD gene correction.
  5. System-Level Analysis of the Effects of RPTPs on Cellular Signaling Networks.
  6. PTEN-restoration abrogates brain colonisation and perivascular niche invasion by melanoma cells.
  7. Phosphoprotein dynamics of interacting T cells and tumor cells by HySic.
  8. Extracellular carriers control lipid-dependent secretion, delivery, and activity of WNT morphogens.
  9. An Optimized Mouse Embryonic Stem Cell Based Reverse Poly-Transfection Technique for Rapid Exploration of Nucleic Acid Ratios.
  10. Production of CRISPR-Cas9 Transgenic Cell Lines for Knocksideways Studies.
  11. A recurrent de novo MAX p.Arg60Gln variant causes a syndromic overgrowth disorder through differential expression of c-Myc target genes.
  12. Highly cooperative chimeric super-SOX induces naive pluripotency across species.
  1. J Biol Chem. 2023 Dec 21. pii: S0021-9258(23)02611-X. [Epub ahead of print] 105583
    Actin-binding protein Profilin1 is an important determinant of cellular phosphoinositide control.
    Morgan M C Ricci, Andrew Orenberg, Lee Ohayon, David Gau, Rachel C Wills, Yongho Bae, Tuhin Das, David Koes, Gerald R V Hammond, Partha Roy.
      Membrane poly-phosphoinositides (PPIs) are lipid-signaling molecules that undergo metabolic turnover and influence a diverse range of cellular functions. PPIs regulate the activity and/or spatial localization of a number of actin-binding proteins (ABPs) through direct interactions; however, it is much less clear whether ABPs could also be an integral part in regulating PPI signaling. In this study, we show that ABP profilin1 (Pfn1) is an important molecular determinant of cellular content of PI(4,5)P2 (the most abundant PPI in cells). In growth-factor (EGF) stimulation setting, Pfn1 depletion does not impact PI(4,5)P2 hydrolysis but enhances PM enrichment of PPIs that are produced downstream of activated PI3-Kinase including PI(3,4,5)P3 and PI(3,4)P2, the latter consistent with increased PM recruitment of SHIP2 (a key enzyme for PI(3,4)P2 biosynthesis). Although Pfn1 binds to PPIs in vitro, our data suggest that Pfn1's affinity to PPIs and PM presence in actual cells, if at all, is negligible, suggesting that Pfn1 is unlikely to directly compete with SHIP2 for binding to PM PPIs. Additionally, we provide evidence for Pfn1's interaction with SHIP2 in cells, and modulation of this interaction upon EGF stimulation, raising an alternative possibility of Pfn1 binding as a potential restrictive mechanism for PM recruitment of SHIP2. In conclusion, our findings challenge the dogma of Pfn1's binding to PM by PPI interaction, uncover a previously unrecognized role of Pfn1 in PI(4,5)P2 homeostasis, and provide a new mechanistic avenue of how an ABP could potentially impact PI3K signaling byproducts in cells through lipid phosphatase control.
    Keywords:  PI(3,4)P(2); PI(3,4,5)P(3); PI(4,5)P(2); Phosphoinositides; Profilin; SHIP2
    DOI:  https://doi.org/10.1016/j.jbc.2023.105583
  2. J Clin Immunol. 2023 Dec 27. 44(1): 34
    Modulating the PI3K Signalling Pathway in Activated PI3K Delta Syndrome: a Clinical Perspective.
    Lucinda J Berglund.
      Activated phosphoinositide-3-kinase (PI3K) δ syndrome (APDS) is an inborn error of immunity characterised by immune dysregulation. Since the discovery of genetic mutations resulting in PI3Kδ overactivation, treatment of APDS patients has begun to focus on modulation of the PI3K pathway in addition to supportive therapies. The mTOR inhibitor sirolimus has been used effectively for some clinical manifestations of this condition, however the arrival of specific PI3Kδ inhibitor leniolisib has shown promising early results and may provide a more targeted approach. This review summarizes key aspects of PI3K pathway biology and discusses potential options for nuanced modulation of the PI3K pathway in APDS from a clinical perspective, highlighting differences from PI3K inhibition in haematological malignancies.
    Keywords:  APDS; Activated PI3K delta syndrome; leniolisib; sirolimus
    DOI:  https://doi.org/10.1007/s10875-023-01626-0
  3. Front Cell Dev Biol. 2023 ;11 1271141
    Signaling plasticity in the integrated stress response.
    Morgane Boone, Francesca Zappa.
      The Integrated Stress Response (ISR) is an essential homeostatic signaling network that controls the cell's biosynthetic capacity. Four ISR sensor kinases detect multiple stressors and relay this information to downstream effectors by phosphorylating a common node: the alpha subunit of the eukaryotic initiation factor eIF2. As a result, general protein synthesis is repressed while select transcripts are preferentially translated, thus remodeling the proteome and transcriptome. Mounting evidence supports a view of the ISR as a dynamic signaling network with multiple modulators and feedback regulatory features that vary across cell and tissue types. Here, we discuss updated views on ISR sensor kinase mechanisms, how the subcellular localization of ISR components impacts signaling, and highlight ISR signaling differences across cells and tissues. Finally, we consider crosstalk between the ISR and other signaling pathways as a determinant of cell health.
    Keywords:  homeostasis; integrated stress response; sensor kinase; signal crosstalk; signal heterogeneity; signaling network; subcellular compartmentalization
    DOI:  https://doi.org/10.3389/fcell.2023.1271141
  4. Mol Ther Methods Clin Dev. 2023 Dec 14. 31 101133
    Rescuing the cytolytic function of APDS1 patient T cells via TALEN-mediated PIK3CD gene correction.
    Lucie Poggi, Loïc Chentout, Sabrina Lizot, Alex Boyne, Alexandre Juillerat, Arianna Moiani, Marine Luka, Francesco Carbone, Mickael Ménager, Marina Cavazzana, Philippe Duchateau, Julien Valton, Sven Kracker.
      Gain-of-function mutations in the PIK3CD gene result in activated phosphoinositide 3-kinase δ syndrome type 1 (APDS1). This syndrome is a life-threatening combined immunodeficiency and today there are neither optimal nor long-term therapeutic solutions for APDS1 patients. Thus, new alternative treatments are highly needed. The aim of the present study is to explore one therapeutic avenue that consists of the correction of the PIK3CD gene through gene editing. Our proof-of-concept shows that TALEN-mediated gene correction of the mutated PIK3CD gene in APDS1 T cells results in normalized phospho-AKT levels in basal and activated conditions. Normalization of PI3K signaling was correlated to restored cytotoxic functions of edited CD8+ T cells. At the transcriptomic level, single-cell RNA sequencing revealed corrected signatures of CD8+ effector memory and CD8+ proliferating T cells. This proof-of-concept study paves the way for the future development of a gene therapy candidate to cure activated phosphoinositide 3-kinase δ syndrome type 1.
    Keywords:  APDS1; PI3K signaling; TALEN; gene editing; serial killing assay; single-cell RNA sequencing
    DOI:  https://doi.org/10.1016/j.omtm.2023.101133
  5. Methods Mol Biol. 2024 ;2743 153-163
    System-Level Analysis of the Effects of RPTPs on Cellular Signaling Networks.
    Jacqueline Gerritsen, Sophie Rizzo, Damien Thévenin, Forest M White.
      Tyrosine phosphorylation regulates signaling network activity downstream of receptor tyrosine kinase (RTK) activation. Receptor protein tyrosine phosphatases (RPTPs) serve to dephosphorylate RTKs and their proximal adaptor proteins, thus serving to modulate RTK activity. While the general function of RPTPs is well understood, the direct and indirect substrates for each RPTP are poorly characterized. Here we describe a method, quantitative phosphotyrosine phosphoproteomics, that enables the identification of specific phosphorylation sites whose phosphorylation levels are altered by the expression and activity of a given RPTP. In a proof-of-concept application, we use this method to highlight several direct or indirect substrate phosphorylation sites for PTPRJ, also known as DEP1, and show their quantitative phosphorylation in the context of wild-type PTPRJ compared to a mutant form of PTPRJ with increased activity, in EGF-stimulated cells. This method is generally applicable to define the signaling network effects of each RPTP in cells or tissues under different physiological conditions.
    Keywords:  Phosphatase activity; Signaling networks; Tyrosine phosphorylation
    DOI:  https://doi.org/10.1007/978-1-0716-3569-8_10
  6. Br J Cancer. 2023 Dec 26.
    PTEN-restoration abrogates brain colonisation and perivascular niche invasion by melanoma cells.
    Sarah Wang, Caroline P Riedstra, Yu Zhang, Swetha Anandh, Andrew C Dudley.
       BACKGROUND: Melanoma brain metastases (MBM) continue to be a significant clinical problem with limited treatment options. Highly invasive melanoma cells migrate along the vasculature and perivascular cells may contribute to residual disease and recurrence. PTEN loss and hyperactivation of AKT occur in MBM; however, a role for PTEN/AKT in perivascular invasion has not been described.
    METHODS: We used in vivo intracranial injections of murine melanoma and bulk RNA sequencing of melanoma cells co-cultured with brain endothelial cells (brECs) to investigate brain colonisation and perivascular invasion.
    RESULTS: We found that PTEN-null melanoma cells were highly efficient at colonising the perivascular niche relative to PTEN-expressing counterparts. PTEN re-expression (PTEN-RE) in melanoma cells significantly reduced brain colonisation and migration along the vasculature. We hypothesised this phenotype was mediated through vascular-induced TGFβ secretion, which drives AKT phosphorylation. Disabling TGFβ signalling in melanoma cells reduced colonisation and perivascular invasion; however, the introduction of constitutively active myristolated-AKT (myrAKT) restored overall tumour size but not perivascular invasion.
    CONCLUSIONS: PTEN loss facilitates perivascular brain colonisation and invasion of melanoma. TGFβ-AKT signalling partially contributes to this phenotype, but further studies are needed to determine the complementary mechanisms that enable melanoma cells to both survive and spread along the brain vasculature.
    DOI:  https://doi.org/10.1038/s41416-023-02530-5
  7. Cell Rep. 2023 Dec 26. pii: S2211-1247(23)01610-8. [Epub ahead of print]43(1): 113598
    Phosphoprotein dynamics of interacting T cells and tumor cells by HySic.
    Sofía Ibáñez-Molero, Joannes T M Pruijs, Alisha Atmopawiro, Fujia Wang, Alexandra M Terry, Maarten Altelaar, Daniel S Peeper, Kelly E Stecker.
      Functional interactions between cytotoxic T cells and tumor cells are central to anti-cancer immunity. However, our understanding of the proteins involved is limited. Here, we present HySic (hybrid quantification of stable isotope labeling by amino acids in cell culture [SILAC]-labeled interacting cells) as a method to quantify protein and phosphorylation dynamics between and within physically interacting cells. Using co-cultured T cells and tumor cells, we directly measure the proteome and phosphoproteome of engaged cells without the need for physical separation. We identify proteins whose abundance or activation status changes upon T cell:tumor cell interaction and validate our method with established signal transduction pathways including interferon γ (IFNγ) and tumor necrosis factor (TNF). Furthermore, we identify the RHO/RAC/PAK1 signaling pathway to be activated upon cell engagement and show that pharmacologic inhibition of PAK1 sensitizes tumor cells to T cell killing. Thus, HySic is a simple method to study rapid protein signaling dynamics in physically interacting cells that is easily extended to other biological systems.
    Keywords:  CP: Cancer; CP: Immunology; T cell; cancer; cell-cell interaction; immunology; phosphoproteomics; proteomics
    DOI:  https://doi.org/10.1016/j.celrep.2023.113598
  8. Dev Cell. 2023 Dec 21. pii: S1534-5807(23)00648-2. [Epub ahead of print]
    Extracellular carriers control lipid-dependent secretion, delivery, and activity of WNT morphogens.
    Taciani de Almeida Magalhaes, Jingjing Liu, Charlene Chan, Kleiton Silva Borges, Jiuchun Zhang, Andrew J Kane, Bradley M Wierbowski, Yunhui Ge, Zhiwen Liu, Prabhath Mannam, Daniel Zeve, Ron Weiss, David T Breault, Pengxiang Huang, Adrian Salic.
      WNT morphogens trigger signaling pathways fundamental for embryogenesis, regeneration, and cancer. WNTs are modified with palmitoleate, which is critical for binding Frizzled (FZD) receptors and activating signaling. However, it is unknown how WNTs are released and spread from cells, given their strong lipid-dependent membrane attachment. We demonstrate that secreted FZD-related proteins and WNT inhibitory factor 1 are WNT carriers, potently releasing lipidated WNTs and forming active soluble complexes. WNT release occurs by direct handoff from the membrane protein WNTLESS to the carriers. In turn, carriers donate WNTs to glypicans and FZDs involved in WNT reception and to the NOTUM hydrolase, which antagonizes WNTs by lipid moiety removal. WNT transfer from carriers to FZDs is greatly facilitated by glypicans that serve as essential co-receptors in Wnt signaling. Thus, an extracellular network of carriers dynamically controls secretion, posttranslational regulation, and delivery of WNT morphogens, with important practical implications for regenerative medicine.
    Keywords:  WNT; extracellular; lipids; morphogen; receptor; signaling
    DOI:  https://doi.org/10.1016/j.devcel.2023.11.027
  9. J Vis Exp. 2023 Dec 08.
    An Optimized Mouse Embryonic Stem Cell Based Reverse Poly-Transfection Technique for Rapid Exploration of Nucleic Acid Ratios.
    Kieran Maheden, Karen Hwang, Ipek Egilmez, Nika Shakiba.
      Due to its relative simplicity and ease of use, transient transfection of mammalian cell lines with nucleic acids has become a mainstay in biomedical research. While most widely used cell lines have robust protocols for transfection in adherent two-dimensional culture, these protocols often do not translate well to less-studied lines or those with atypical, hard-to-transfect morphologies. Using mouse pluripotent stem cells grown in 2i/LIF media, a widely used culture model for regenerative medicine, this method outlines an optimized, rapid reverse transfection protocol capable of achieving higher transfection efficiency. Leveraging this protocol, a three-plasmid poly-transfection is performed, taking advantage of the higher-than-normal efficiency in plasmid delivery to study an expanded range of plasmid stoichiometry. This reverse poly-transfection protocol allows for a one-pot experimental method, enabling users to optimize plasmid ratios in a single well, rather than across several co-transfections. By facilitating the rapid exploration of the effect of DNA stoichiometry on the overall function of delivered genetic circuits, this protocol minimizes the time and cost of embryonic stem cell transfection.
    DOI:  https://doi.org/10.3791/65766
  10. Curr Protoc. 2023 Dec;3(12): e965
    Production of CRISPR-Cas9 Transgenic Cell Lines for Knocksideways Studies.
    Michael Wagenbach, Juan Jesus Vicente, Wren Wagenbach, Linda Wordeman.
      Protein activity is generally functionally integrated and spatially restricted to key locations within the cell. Knocksideways experiments allow researchers to rapidly move proteins to alternate or ectopic regions of the cell and assess the resultant cellular response. Briefly, individual proteins to be tested using this approach must be modified with moieties that dimerize under treatment with rapamycin to promote the experimental spatial relocalizations. CRISPR technology enables researchers to engineer modified protein directly in cells while preserving proper protein levels because the engineered protein will be expressed from endogenous promoters. Here we provide straightforward instructions to engineer tagged, rapamycin-relocalizable proteins in cells. The protocol is described in the context of our work with the microtubule depolymerizer MCAK/Kif2C, but it is easily adaptable to other genes and alternate tags such as degrons, optogenetic constructs, and other experimentally useful modifications. Off-target effects are minimized by testing for the most efficient target site using a split-GFP construct. This protocol involves no proprietary kits, only plasmids available from repositories (such as addgene.org). Validation, relocalization, and some example novel discoveries obtained working with endogenous protein levels are described. A graduate student with access to a fluorescence microscope should be able to prepare engineered cells with spatially controllable endogenous protein using this protocol. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Choosing a target site for gene modification Basic Protocol 2: Design of gRNA(s) for targeted gene modification Basic Protocol 3: Split-GFP test for target efficiency Basic Protocol 4: Design of the recombination template and analytical primers Support Protocol 1: Design of primers for analytical PCR Basic Protocol 5: Transfection, isolation, and validation of engineered cells Support Protocol 2: Stable transfection of engineered cells with binding partners.
    Keywords:  CRISPR; Cas9; GFP; Kif2C; knocksideways
    DOI:  https://doi.org/10.1002/cpz1.965
  11. Am J Hum Genet. 2023 Dec 14. pii: S0002-9297(23)00428-7. [Epub ahead of print]
    A recurrent de novo MAX p.Arg60Gln variant causes a syndromic overgrowth disorder through differential expression of c-Myc target genes.
    Erica L Harris, Vincent Roy, Martin Montagne, Ailsa M S Rose, Helen Livesey, Margot R F Reijnders, Emma Hobson, Francis H Sansbury, Marjolein H Willemsen, Rolph Pfundt, Daniel Warren, Vernon Long, Ian M Carr, Han G Brunner, Eamonn G Sheridan, Helen V Firth, Pierre Lavigne, James A Poulter.
      Cyclin D2 (CCND2) stabilization underpins a range of macrocephaly-associated disorders through mutation of CCND2 or activating mutations in upstream genes encoding PI3K-AKT pathway components. Here, we describe three individuals with overlapping macrocephaly-associated phenotypes who carry the same recurrent de novo c.179G>A (p.Arg60Gln) variant in Myc-associated factor X (MAX). The mutation, located in the b-HLH-LZ domain, causes increased intracellular CCND2 through increased transcription but it does not cause stabilization of CCND2. We show that the purified b-HLH-LZ domain of MAXArg60Gln (Max∗Arg60Gln) binds its target E-box sequence with a lower apparent affinity. This leads to a more efficient heterodimerization with c-Myc resulting in an increase in transcriptional activity of c-Myc in individuals carrying this mutation. The recent development of Omomyc-CPP, a cell-penetrating b-HLH-LZ-domain c-Myc inhibitor, provides a possible therapeutic option for MAXArg60Gln individuals, and others carrying similar germline mutations resulting in dysregulated transcriptional c-Myc activity.
    Keywords:  CCND2; MAX; MYC; b-HLH-LZ; macrocephaly; polydactyly
    DOI:  https://doi.org/10.1016/j.ajhg.2023.11.010
  12. Cell Stem Cell. 2023 Dec 20. pii: S1934-5909(23)00402-2. [Epub ahead of print]
    Highly cooperative chimeric super-SOX induces naive pluripotency across species.
    Caitlin M MacCarthy, Guangming Wu, Vikas Malik, Yotam Menuchin-Lasowski, Taras Velychko, Gal Keshet, Rui Fan, Ivan Bedzhov, George M Church, Ralf Jauch, Vlad Cojocaru, Hans R Schöler, Sergiy Velychko.
      Our understanding of pluripotency remains limited: iPSC generation has only been established for a few model species, pluripotent stem cell lines exhibit inconsistent developmental potential, and germline transmission has only been demonstrated for mice and rats. By swapping structural elements between Sox2 and Sox17, we built a chimeric super-SOX factor, Sox2-17, that enhanced iPSC generation in five tested species: mouse, human, cynomolgus monkey, cow, and pig. A swap of alanine to valine at the interface between Sox2 and Oct4 delivered a gain of function by stabilizing Sox2/Oct4 dimerization on DNA, enabling generation of high-quality OSKM iPSCs capable of supporting the development of healthy all-iPSC mice. Sox2/Oct4 dimerization emerged as the core driver of naive pluripotency with its levels diminished upon priming. Transient overexpression of the SK cocktail (Sox+Klf4) restored the dimerization and boosted the developmental potential of pluripotent stem cells across species, providing a universal method for naive reset in mammals.
    Keywords:  Oct4; POU linker; Sox17; Sox2; Sox2/Oct4 heterodimer structure; bovine; developmental potential; engineered transcription factor; human; iPSC; mouse; naive pluripotency; non-human primate; porcine; reprogramming; reset; super-SOX; tetraploid complementation
    DOI:  https://doi.org/10.1016/j.stem.2023.11.010
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