bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2022–10–02
24 papers selected by
Ralitsa Radostinova Madsen, University College London
  1. PtdIns(3,4)P2, Lamellipodin, and VASP coordinate actin dynamics during phagocytosis in macrophages.
  2. Clinical and genetic analyses of patients with lateralized overgrowth.
  3. Loss of the Fbw7 tumor suppressor rewires cholesterol metabolism in cancer cells leading to activation of the PI3K-AKT signalling axis.
  4. Somatic variation in normal tissues: friend or foe of cancer early detection?
  5. Analysis of the Human Protein Atlas Weakly Supervised Single-Cell Classification competition.
  6. Inferring kinase activity from phosphoproteomic data: Tool comparison and recent applications.
  7. IBCL-271 Primary Efficacy and Safety Analysis of a Global Phase II Study of Zandelisib Administered by Intermittent Dosing (ID) in Patients With Relapsed or Refractory (R/R) Follicular Lymphoma (FL): The TIDAL Study.
  8. Sustained Aurora kinase B expression confers resistance to PI3K inhibition in head and neck squamous carcinoma.
  9. Autocrine FGF1 signaling promotes glucose uptake in adipocytes.
  10. Comprehensive targeted next-generation sequencing in patients with slow-flow vascular malformations.
  11. Activation of the human insulin receptor by non-insulin-related peptides.
  12. ERK signaling dynamics: Lights, camera, transduction.
  13. Targeting p85β nuclear translocation for the tumors with PIK3CA helical domain mutations.
  14. PI3Kβ-regulated β-catenin mediates EZH2 removal from promoters controlling primed human ESC stemness and primitive streak gene expression.
  15. Quantitative Cell Proteomic Atlas: Pathway-Scale Targeted Mass Spectrometry for High-Resolution Functional Profiling of Cell Signaling.
  16. The VersaLive platform enables microfluidic mammalian cell culture for versatile applications.
  17. The Grb2 splice variant, Grb3-3, is a negative regulator of RAS activation.
  18. Highly sensitive single-cell chromatin accessibility assay and transcriptome coassay with METATAC.
  19. Cell Taxonomy: a curated repository of cell types with multifaceted characterization.
  20. Quantification of Genome Editing and Transcriptional Control Capabilities Reveals Hierarchies among Diverse CRISPR/Cas Systems in Human Cells.
  21. qPTM: an updated database for PTM dynamics in human, mouse, rat and yeast.
  22. Experimental reproducibility limits the correlation between mRNA and protein abundances in tumor proteomic profiles.
  23. Determining and interpreting protein lifetimes in mammalian tissues.
  24. Metabolite-driven antitumor immunity.
  1. J Cell Biol. 2022 Nov 07. pii: e202207042. [Epub ahead of print]221(11):
    PtdIns(3,4)P2, Lamellipodin, and VASP coordinate actin dynamics during phagocytosis in macrophages.
    Fernando Montaño-Rendón, Glenn F W Walpole, Matthias Krause, Gerald R V Hammond, Sergio Grinstein, Gregory D Fairn.
      Phosphoinositides are pivotal regulators of vesicular traffic and signaling during phagocytosis. Phagosome formation, the initial step of the process, is characterized by local membrane remodeling and reorganization of the actin cytoskeleton that leads to formation of the pseudopods that drive particle engulfment. Using genetically encoded fluorescent probes, we found that upon particle engagement a localized pool of PtdIns(3,4)P2 is generated by the sequential activities of class I phosphoinositide 3-kinases and phosphoinositide 5-phosphatases. Depletion of this locally generated pool of PtdIns(3,4)P2 blocks pseudopod progression and ultimately phagocytosis. We show that the PtdIns(3,4)P2 effector Lamellipodin (Lpd) is recruited to nascent phagosomes by PtdIns(3,4)P2. Furthermore, we show that silencing of Lpd inhibits phagocytosis and produces aberrant pseudopodia with disorganized actin filaments. Finally, vasodilator-stimulated phosphoprotein (VASP) was identified as a key actin-regulatory protein mediating phagosome formation downstream of Lpd. Mechanistically, our findings imply that a pathway involving PtdIns(3,4)P2, Lpd, and VASP mediates phagocytosis at the stage of particle engulfment.
    DOI:  https://doi.org/10.1083/jcb.202207042
  2. BMC Med Genomics. 2022 Sep 30. 15(1): 206
    Clinical and genetic analyses of patients with lateralized overgrowth.
    Yoon-Myung Kim, Yena Lee, Yunha Choi, In Hee Choi, Sun Hee Heo, Jung Min Choi, Hyo-Sang Do, Ja-Hyun Jang, Mi-Sun Yum, Han-Wook Yoo, Beom Hee Lee.
       BACKGROUND: The genetic features and treatment strategies of lateralized overgrowth have been elusive. We performed this study to analyze the genetic characteristics and treatment results of propranolol- or alpelisib-treated patients with lateralized overgrowth.
    METHODS: Fifteen patients with lateralized overgrowth were involved. Clinical characteristics and whole-body magnetic resonance imaging (WB-MRI) findings were evaluated. Targeted exome sequencing with a gene panel of affected tissue and peripheral white blood cells was performed. Propranolol was administered and treatment results were evaluated. The PIK3CA inhibitor alpelisib was prescribed via a managed access program.
    RESULTS: The identified mutations were PIK3CA (n = 7), KRAS (n = 2), PTEN (n = 1), MAP2K3 (n = 1), GNAQ (n = 1), TBC1D4 (n = 1), and TEK (n = 1). Propranolol was prescribed in 12 patients, and 7 experienced mild improvement of symptoms. Alpelisib was prescribed in two patients with a PIK3CA mutation, and the reduction of proliferated masses after 1 year of treatment was proved by WB-MRI.
    CONCLUSIONS: Targeted exome sequencing identified various genetic features of lateralized overgrowth. Propranolol could be applied as an adjuvant therapy for reducing vascular symptoms, but a PIK3CA inhibitor would be the primary therapeutic strategy for PIK3CA-related overgrowth syndrome.
    Keywords:  Alpelisib; Lateralized overgrowth; PIK3CA-related segmental overgrowth syndrome; Targeted exome sequencing
    DOI:  https://doi.org/10.1186/s12920-022-01362-1
  3. Front Oncol. 2022 ;12 990672
    Loss of the Fbw7 tumor suppressor rewires cholesterol metabolism in cancer cells leading to activation of the PI3K-AKT signalling axis.
    Maria T Bengoechea-Alonso, Arwa Aldaalis, Johan Ericsson.
      The sterol regulatory-element binding proteins (SREBPs) are transcription factors controlling cholesterol and fatty acid synthesis and metabolism. There are three SREBP proteins, SREBP1a, SREBP1c and SREBP2, with SREBP1a being the strongest transcription factor. The expression of SREBP1a is restricted to rapidly proliferating cells, including cancer cells. The SREBP proteins are translated as large, inactive precursors bound to the endoplasmic reticulum (ER) membranes. These precursors undergo a two-step cleavage process that releases the amino terminal domains of the proteins, which translocate to the nucleus and function as transcription factors. The nuclear forms of the SREBPs are rapidly degraded by the ubiquitin-proteasome system in a manner dependent on the Fbw7 ubiquitin ligase. Consequently, inactivation of Fbw7 results in the stabilization of active SREBP1 and SREBP2 and enhanced expression of target genes. We report that the inactivation of Fbw7 in cancer cells blocks the proteolytic maturation of SREBP2. The same is true in cells expressing a cancer-specific loss-of-function Fbw7 protein. Interestingly, the activation of SREBP2 is restored in response to cholesterol depletion, suggesting that Fbw7-deficient cells accumulate cholesterol. Importantly, inactivation of SREBP1 in Fbw7-deficient cells also restores the cholesterol-dependent regulation of SREBP2, suggesting that the stabilization of active SREBP1 molecules could be responsible for the blunted activation of SREBP2 in Fbw7-deficient cancer cells. We suggest that this could be an important negative feedback loop in cancer cells with Fbw7 loss-of-function mutations to protect these cells from the accumulation of toxic levels of cholesterol and/or cholesterol metabolites. Surprisingly, we also found that the inactivation of Fbw7 resulted in the activation of AKT. Importantly, the activation of AKT was dependent on SREBP1 and on the accumulation of cholesterol. Thus, we suggest that the loss of Fbw7 rewires lipid metabolism in cancer cells to support cell proliferation and survival.
    Keywords:  AKT; Fbw7; PI3K; SREBP; cancer; cholesterol
    DOI:  https://doi.org/10.3389/fonc.2022.990672
  4. Ann Oncol. 2022 Sep 23. pii: S0923-7534(22)04148-5. [Epub ahead of print]
    Somatic variation in normal tissues: friend or foe of cancer early detection?
    A Acha-Sagredo, P Ganguli, F D Ciccarelli.
       BACKGROUND: Seemingly normal tissues progressively become populated by mutant clones over time. Most of these clones bear mutations in well-known cancer genes but only rarely do they transform into cancer. This poses questions on what triggers cancer initiation and what implications somatic variation has for cancer early detection.
    DESIGN: We analysed recent mutational screens of healthy and cancer-free diseased tissues to compare somatic drivers and the causes of somatic variation across tissues. We then reviewed the mechanisms of clonal expansion and their relationships with age and diseases other than cancer. We finally discussed the relevance of somatic variation for cancer initiation and how it can help or hinder cancer detection and prevention.
    RESULTS: The extent of somatic variation is highly variable across tissues and depends on intrinsic features, such as tissue architecture and turnover, as well as the exposure to endogenous and exogenous insults. Most somatic mutations driving clonal expansion are tissue-specific and inactivate tumor suppressor genes involved in chromatin modification and cell growth signaling. Some of these genes are more frequently mutated in normal tissues than cancer, indicating a context-dependent cancer promoting or protective role. Mutant clones can persist over a long time or disappear rapidly, suggesting that their fitness depends on the dynamic equilibrium with the environment. The disruption of this equilibrium is likely responsible for their transformation into malignant clones and knowing what triggers this process is key for cancer prevention and early detection. Somatic variation should be considered in liquid biopsy, where it may contribute cancer-independent mutations, and in the identification of cancer drivers, since not all mutated genes favoring clonal expansion also drive tumorigenesis.
    CONCLUSIONS: Somatic variation and the factors governing homeostasis of normal tissues should be taken into account when devising strategies for cancer prevention and early detection.
    Keywords:  Somatic evolution; cancer early detection; cancer initiation; clone selection; driver gene; healthy tissues
    DOI:  https://doi.org/10.1016/j.annonc.2022.09.156
  5. Nat Methods. 2022 Sep 29.
    Analysis of the Human Protein Atlas Weakly Supervised Single-Cell Classification competition.
    Trang Le, Casper F Winsnes, Ulrika Axelsson, Hao Xu, Jayasankar Mohanakrishnan Kaimal, Diana Mahdessian, Shubin Dai, Ilya S Makarov, Vladislav Ostankovich, Yang Xu, Eric Benhamou, Christof Henkel, Roman A Solovyev, Nikola Banić, Vito Bošnjak, Ana Bošnjak, Andrija Miličević, Wei Ouyang, Emma Lundberg.
      While spatial proteomics by fluorescence imaging has quickly become an essential discovery tool for researchers, fast and scalable methods to classify and embed single-cell protein distributions in such images are lacking. Here, we present the design and analysis of the results from the competition Human Protein Atlas - Single-Cell Classification hosted on the Kaggle platform. This represents a crowd-sourced competition to develop machine learning models trained on limited annotations to label single-cell protein patterns in fluorescent images. The particular challenges of this competition include class imbalance, weak labels and multi-label classification, prompting competitors to apply a wide range of approaches in their solutions. The winning models serve as the first subcellular omics tools that can annotate single-cell locations, extract single-cell features and capture cellular dynamics.
    DOI:  https://doi.org/10.1038/s41592-022-01606-z
  6. Mass Spectrom Rev. 2022 Sep 26. e21808
    Inferring kinase activity from phosphoproteomic data: Tool comparison and recent applications.
    Sander R Piersma, Andrea Valles-Marti, Frank Rolfs, Thang V Pham, Alex A Henneman, Connie R Jiménez.
      Aberrant cellular signaling pathways are a hallmark of cancer and other diseases. One of the most important signaling mechanisms involves protein phosphorylation/dephosphorylation. Protein phosphorylation is catalyzed by protein kinases, and over 530 protein kinases have been identified in the human genome. Aberrant kinase activity is one of the drivers of tumorigenesis and cancer progression and results in altered phosphorylation abundance of downstream substrates. Upstream kinase activity can be inferred from the global collection of phosphorylated substrates. Mass spectrometry-based phosphoproteomic experiments nowadays routinely allow identification and quantitation of >10k phosphosites per biological sample. This substrate phosphorylation footprint can be used to infer upstream kinase activities using tools like Kinase Substrate Enrichment Analysis (KSEA), Posttranslational Modification Substrate Enrichment Analysis (PTM-SEA), and Integrative Inferred Kinase Activity Analysis (INKA). Since the topic of kinase activity inference is very active with many new approaches reported in the past 3 years, we would like to give an overview of the field. In this review, an inventory of kinase activity inference tools, their underlying algorithms, statistical frameworks, kinase-substrate databases, and user-friendliness is presented. The most widely-used tools are compared in-depth. Subsequently, recent applications of the tools are described focusing on clinical tissues and hematological samples. Two main application areas for kinase activity inference tools can be discerned. (1) Maximal biological insights can be obtained from large data sets with group comparisons using multiple complementary tools (e.g., PTM-SEA and KSEA or INKA). (2) In the oncology context where personalized treatment requires analysis of single samples, INKA for example, has emerged as tool that can prioritize actionable kinases for targeted inhibition.
    DOI:  https://doi.org/10.1002/mas.21808
  7. Clin Lymphoma Myeloma Leuk. 2022 Oct;pii: S2152-2650(22)01555-5. [Epub ahead of print]22 Suppl 2 S387-S388
    IBCL-271 Primary Efficacy and Safety Analysis of a Global Phase II Study of Zandelisib Administered by Intermittent Dosing (ID) in Patients With Relapsed or Refractory (R/R) Follicular Lymphoma (FL): The TIDAL Study.
    Tycel Phillips, Wojciech Jurczak, Vincent Ribrag, Kim Linton, Graham P Collin, Javier Lopéz-Jimenéz, Nishitha Reddy, Andrea Mengarelli, Gerardo Musuraca, Oonagh Sheehy, Weiming Xu, Michel Azoulay, Richard G Ghalie, Pier Luigi Zinzani, Andrew D Zelenetz.
       CONTEXT: Zandelisib is a selective PI3Kδ inhibitor administered orally at 60 mg once daily (QD) for 2 cycles (response induction), then intermittent dosing (ID) on days 1-7 of subsequent 28-day cycles for maintenance, while potentially enabling regulatory T-cell recovery to reduce risk of immune adverse events (irAEs) seen with continuous PI3Kδ inhibition. In a phase Ib study of zandelisib in 37 R/R FL patients, the overall response rate (ORR) was 87% (78% single agent; 95% with rituximab), with only 8% discontinuations due to irAEs (Pagel ICML 2021).
    OBJECTIVE: Topline results from the fully enrolled FL population from TIDAL, a global phase II study evaluating zandelisib in R/R indolent lymphomas (NCT03768505).
    PATIENTS: Age ≥18y with FL Grade I-IIIA, progressive disease after ≥2 prior therapies, and no prior PI3K inhibitor. Consent required. FL sample size (planned): 120 patients. Primary efficacy population (PEP): first 91 patients treated.
    INTERVENTION: Zandelisib 60 mg QD for 2 cycles, followed by ID during cycle 3+.
    MAIN OUTCOME MEASURE: IRC-assessed ORR (Lugano criteria) after a minimum 6-month follow-up.
    RESULTS: 91 FL patients in PEP (of 121 enrolled): median 3 prior therapies (range, 2-8), 21 (23%) prior stem cell transplant, 42 (46%) refractory to last therapy, 31 (34%) tumors ≥5 cm, 51 (56%) POD24. ORR was 70.3% (64/91; 95% CI: 59.8%, 79.5%) and complete response (CR) rate was 35.2% (32/91; 95% CI: 25.4%, 45.9%). Responses occurred early: 87.5% (56/91) occurred at end of Cycle 2, 75% (24/91) of CRs at end of Cycle 4. Data still immature for accurate duration of response (DOR) estimation. With median follow-up of 9.4 months (range, 0.8-24) for all 121 patients, 12 (9.9%) discontinued due to any treatment-related AE. Grade 3 AEs of special interest (AESI) included diarrhea (6/121; 5%), colitis (2/121; 1.7%), rash (4/121; 3.3%), stomatitis (3/121; 2.5%), and AST and ALT elevation and non-infectious pneumonitis (1/121 each; 0.8%). Most Grade 3 AESIs (15 [83%]) occurred during daily dosing (cycles 1-3).
    CONCLUSIONS: Zandelisib on ID led to high ORR and CR rates in heavily pretreated FL patients and was associated with a low rate of grade 3 AESI and discontinuations due to treatment-related AEs.
    Keywords:  IBCL; PI3Kδ inhibitor; Phase II; TIDAL; clinical trial; follicular lymphoma; zandelisib
    DOI:  https://doi.org/10.1016/S2152-2650(22)01555-5
  8. Cancer Res. 2022 Sep 28. pii: CAN-22-1175. [Epub ahead of print]
    Sustained Aurora kinase B expression confers resistance to PI3K inhibition in head and neck squamous carcinoma.
    Pooja A Shah, Vaishnavi Sambandam, Anne M Fernandez, Hongyun Zhao, Tuhina Mazumdar, Li Shen, Qi Wang, Kazi M Ahmed, Soma Ghosh, Mitchell J Frederick, Jing Wang, Faye M Johnson.
      Tumor suppressor mutations in head and neck squamous cell carcinoma (HNSCC) dominate the genomic landscape, hindering the development of effective targeted therapies. Truncating and missense mutations in NOTCH1 are frequent in HNSCC, and inhibition of PI3K can selectively target NOTCH1 mutant (NOTCH1MUT) HNSCC cells . In this study, we identify several proteins that are differentially regulated in HNSCC cells after PI3K inhibition based on NOTCH1 mutation status. Expression of Aurora kinase B (Aurora B), AKT, and PDK1 following PI3K inhibition was significantly lower in NOTCH1MUT cell lines than in NOTCH1WT cells or NOTCH1MUT cells with acquired resistance to PI3K inhibition. Combined inhibition of PI3K and Aurora B was synergistic, enhancing apoptosis in vitro and leading to durable tumor regression in vivo. Overexpression of Aurora B in NOTCH1MUT HNSCC cells led to resistance to PI3K inhibition, while Aurora B knockdown increased sensitivity of NOTCH1WT cells. Additionally, overexpression of Aurora B in NOTCH1MUT HNSCC cells increased total protein levels of AKT and PDK1. AKT depletion in NOTCH1WT cells and overexpression in NOTCH1MUT cells similarly altered sensitivity to PI3K inhibition, and manipulation of AKT levels affected PDK1 but not Aurora B levels. These data define a novel pathway in which Aurora B upregulates AKT that subsequently increases PDK1 selectively in NOTCH1MUT cells to mediate HNSCC survival in response to PI3K inhibition. These findings may lead to an effective therapeutic approach for HNSCC with NOTCH1 mutations while sparing normal cells.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-22-1175
  9. Proc Natl Acad Sci U S A. 2022 Oct 04. 119(40): e2122382119
    Autocrine FGF1 signaling promotes glucose uptake in adipocytes.
    Vera J M Nies, Dicky Struik, Sihao Liu, Weilin Liu, Janine K Kruit, Michael Downes, Tim van Zutphen, Henkjan J Verkade, Ronald M Evans, Johan W Jonker.
      Fibroblast growth factor 1 (FGF1) is an autocrine growth factor released from adipose tissue during over-nutrition or fasting to feeding transition. While local actions underlie the majority of FGF1's anti-diabetic functions, the molecular mechanisms downstream of adipose FGF receptor signaling are unclear. We investigated the effects of FGF1 on glucose uptake and its underlying mechanism in murine 3T3-L1 adipocytes and in ex vivo adipose explants from mice. FGF1 increased glucose uptake in 3T3-L1 adipocytes and epididymal WAT (eWAT) and inguinal WAT (iWAT). Conversely, glucose uptake was reduced in eWAT and iWAT of FGF1 knockout mice. We show that FGF1 acutely increased adipocyte glucose uptake via activation of the insulin-sensitive glucose transporter GLUT4, involving dynamic crosstalk between the MEK1/2 and Akt signaling proteins. Prolonged exposure to FGF1 stimulated adipocyte glucose uptake by MEK1/2-dependent transcription of the basal glucose transporter GLUT1. We have thus identified an alternative pathway to stimulate glucose uptake in adipocytes, independent from insulin, which could open new avenues for treating patients with type 2 diabetes.
    Keywords:  FGF1; adipocytes; fibroblast growth factors; glucose metabolism; insulin
    DOI:  https://doi.org/10.1073/pnas.2122382119
  10. J Hum Genet. 2022 Sep 29.
    Comprehensive targeted next-generation sequencing in patients with slow-flow vascular malformations.
    Akifumi Nozawa, Akihiro Fujino, Shunsuke Yuzuriha, Souichi Suenobu, Aiko Kato, Fumiaki Shimizu, Noriko Aramaki-Hattori, Kanako Kuniyeda, Kazuya Sakaguchi, Hidenori Ohnishi, Yoko Aoki, Michio Ozeki.
      Recent studies have shown that the PI3K signaling pathway plays an important role in the pathogenesis of slow-flow vascular malformations (SFVMs). Analysis of genetic mutations has advanced our understanding of the mechanisms involved in SFVM pathogenesis and may identify new therapeutic targets. We screened for somatic variants in a cohort of patients with SFVMs using targeted next-generation sequencing. Targeted next-generation sequencing of 29 candidate genes associated with vascular anomalies or with the PI3K signaling pathway was performed on affected tissues from patients with SFVMs. Fifty-nine patients with SFVMs (venous malformations n  =  21, lymphatic malformations n  =  27, lymphatic venous malformations n  =  1, and Klippel-Trenaunay syndrome n  =  10) were included in the study. TEK and PIK3CA were the most commonly mutated genes in the study. We detected eight TEK pathogenic variants in 10 samples (16.9%) and three PIK3CA pathogenic variants in 28 samples (47.5%). In total, 37 of 59 patients (62.7%) with SFVMs harbored pathogenic variants in these three genes involved in the PI3K signaling pathway. Inhibitors of this pathway may prove useful as molecular targeted therapies for SFVMs.
    DOI:  https://doi.org/10.1038/s10038-022-01081-6
  11. Nat Commun. 2022 Sep 28. 13(1): 5695
    Activation of the human insulin receptor by non-insulin-related peptides.
    Nicholas S Kirk, Qi Chen, Yingzhe Ginger Wu, Anastasia L Asante, Haitao Hu, Juan F Espinosa, Francisco Martínez-Olid, Mai B Margetts, Faiz A Mohammed, Vladislav V Kiselyov, David G Barrett, Michael C Lawrence.
      The human insulin receptor signalling system plays a critical role in glucose homeostasis. Insulin binding brings about extensive conformational change in the receptor extracellular region that in turn effects trans-activation of the intracellular tyrosine kinase domains and downstream signalling. Of particular therapeutic interest is whether insulin receptor signalling can be replicated by molecules other than insulin. Here, we present single-particle cryoEM structures that show how a 33-mer polypeptide unrelated to insulin can cross-link two sites on the receptor surface and direct the receptor into a signalling-active conformation. The 33-mer polypeptide engages the receptor by two helical binding motifs that are each potentially mimicable by small molecules. The resultant conformation of the receptor is distinct from-but related to-those in extant three-dimensional structures of the insulin-complexed receptor. Our findings thus illuminate unexplored pathways for controlling the signalling of the insulin receptor as well as opportunities for development of insulin mimetics.
    DOI:  https://doi.org/10.1038/s41467-022-33315-8
  12. Dev Cell. 2022 Sep 26. pii: S1534-5807(22)00631-1. [Epub ahead of print]57(18): 2151-2152
    ERK signaling dynamics: Lights, camera, transduction.
    Abhineet Ram, John G Albeck.
      Three-dimensional mammary epithelial acini are a model for understanding how microenvironment-driven signaling coordinates cell behavior and tissue morphogenesis. In this issue of Developmental Cell, Ender et al. use live-cell imaging to capture dynamic spatiotemporal patterns of ERK activity that instruct cell migration and survival fates in developing acini.
    DOI:  https://doi.org/10.1016/j.devcel.2022.09.001
  13. Genes Dis. 2022 Nov;9(6): 1391-1393
    Targeting p85β nuclear translocation for the tumors with PIK3CA helical domain mutations.
    Baoyu He, Yujun Hao.
      
    Keywords:  EZH2; Helical domain mutation; PIK3CA; USP7; p85β
    DOI:  https://doi.org/10.1016/j.gendis.2022.05.018
  14. Stem Cell Reports. 2022 Sep 15. pii: S2213-6711(22)00425-8. [Epub ahead of print]
    PI3Kβ-regulated β-catenin mediates EZH2 removal from promoters controlling primed human ESC stemness and primitive streak gene expression.
    Sudhanshu Yadav, Antonio Garrido, M Carmen Hernández, Juan C Oliveros, Vicente Pérez-García, Mario F Fraga, Ana C Carrera.
      The mechanism governing the transition of human embryonic stem cells (hESCs) toward differentiated cells is only partially understood. To explore this transition, the activity and expression of the ubiquitous phosphatidylinositol 3-kinase (PI3Kα and PI3Kβ) were modulated in primed hESCs. The study reports a pathway that dismantles the restraint imposed by the EZH2 polycomb repressor on an essential stemness gene, NODAL, and on transcription factors required to trigger primitive streak formation. The primitive streak is the site where gastrulation begins to give rise to the three embryonic cell layers from which all human tissues derive. The pathway involves a PI3Kβ non-catalytic action that controls nuclear/active RAC1 levels, activation of JNK (Jun N-terminal kinase) and nuclear β-catenin accumulation. β-Catenin deposition at promoters triggers release of the EZH2 repressor, permitting stemness maintenance (through control of NODAL) and correct differentiation by allowing primitive streak master gene expression. PI3Kβ epigenetic control of EZH2/β-catenin might be modulated to direct stem cell differentiation.
    Keywords:  EZH2; PI3Kβ; Stemness/differentiation transition; clinical hESC differentiation for regeneration medicine; epigenetics; human stem cells; polycomb repression release; primitive streak; stemness; β-catenin
    DOI:  https://doi.org/10.1016/j.stemcr.2022.09.003
  15. J Proteome Res. 2022 Sep 26.
    Quantitative Cell Proteomic Atlas: Pathway-Scale Targeted Mass Spectrometry for High-Resolution Functional Profiling of Cell Signaling.
    Paolo Cifani, Alex Kentsis.
      In spite of extensive studies of cellular signaling, many fundamental processes such as pathway integration, cross-talk, and feedback remain poorly understood. To enable integrated and quantitative measurements of cellular biochemical activities, we have developed the Quantitative Cell Proteomics Atlas (QCPA). QCPA consists of panels of targeted mass spectrometry assays to determine the abundance and stoichiometry of regulatory post-translational modifications of sentinel proteins from most known physiologic and pathogenic signaling pathways in human cells. QCPA currently profiles 1 913 peptides from 469 effectors of cell surface signaling, apoptosis, stress response, gene expression, quiescence, and proliferation. For each protein, QCPA includes triplets of isotopically labeled peptides covering known post-translational regulatory sites to determine their stoichiometries and unmodified protein regions to measure total protein abundance. The QCPA framework incorporates analytes to control for technical variability of sample preparation and mass spectrometric analysis, including TrypQuant, a synthetic substrate for accurate quantification of proteolysis efficiency for proteins containing chemically modified residues. The ability to precisely and accurately quantify most known signaling pathways should enable improved chemoproteomic approaches for the comprehensive analysis of cell signaling and clinical proteomics of diagnostic specimens. QCPA is openly available at https://qcpa.mskcc.org.
    Keywords:  PTM stoichiometry; biochemical regulation; cancer; open access; phosphorylation; post-translational modification; targeted mass spectrometry
    DOI:  https://doi.org/10.1021/acs.jproteome.2c00223
  16. Commun Biol. 2022 Sep 29. 5(1): 1034
    The VersaLive platform enables microfluidic mammalian cell culture for versatile applications.
    Giovanni Marco Nocera, Gaetano Viscido, Stefania Criscuolo, Simona Brillante, Fabrizia Carbone, Leopoldo Staiano, Sabrina Carrella, Diego di Bernardo.
      Microfluidic-based cell culture allows for precise spatio-temporal regulation of microenvironment, live cell imaging and better recapitulation of physiological conditions, while minimizing reagents' consumption. Despite their usefulness, most microfluidic systems are designed with one specific application in mind and usually require specialized equipment and expertise for their operation. All these requirements prevent microfluidic-based cell culture to be widely adopted. Here, we designed and implemented a versatile and easy-to-use perfusion cell culture microfluidic platform for multiple applications (VersaLive) requiring only standard pipettes. Here, we showcase the multiple uses of VersaLive (e.g., time-lapse live cell imaging, immunostaining, cell recovery, cell lysis, plasmid transfection) in mammalian cell lines and primary cells. VersaLive could replace standard cell culture formats in several applications, thus decreasing costs and increasing reproducibility across laboratories. The layout, documentation and protocols are open-source and available online at https://versalive.tigem.it/ .
    DOI:  https://doi.org/10.1038/s42003-022-03976-8
  17. Commun Biol. 2022 Sep 28. 5(1): 1029
    The Grb2 splice variant, Grb3-3, is a negative regulator of RAS activation.
    Caroline Seiler, Amy K Stainthorp, Sophie Ketchen, Christopher M Jones, Kate Marks, Philip Quirke, John E Ladbury.
      Activation of RAS is crucial in driving cellular outcomes including proliferation, differentiation, migration and apoptosis via the MAPK pathway. This is initiated on recruitment of Grb2, as part of a Grb2-Sos complex, to an up-regulated receptor tyrosine kinase (RTK), enabling subsequent interaction of Sos with the plasma membrane-localised RAS. Aberrant regulation at this convergence point for RTKs in MAPK signalling is a key driver of multiple cancers. Splicing of the GRB2 gene produces a deletion variant, Grb3-3, that is incapable of binding to RTKs. We show that, despite maintaining the ability to bind to Sos, the Grb3-3-Sos complex remains in the cytoplasm, unable to engage with RAS. Competition between Grb2 and Grb3-3 for binding to C-terminal proline-rich sequences on Sos modulates MAPK signalling. Additionally, we demonstrate that splicing is regulated by heterogenous nuclear riboproteins C1/C2, and that normal and malignant colon tissue show differential Grb3-3 expression.
    DOI:  https://doi.org/10.1038/s42003-022-03985-7
  18. Proc Natl Acad Sci U S A. 2022 Oct 04. 119(40): e2206450119
    Highly sensitive single-cell chromatin accessibility assay and transcriptome coassay with METATAC.
    Honggui Wu, Xiang Li, Fanchong Jian, Ayijiang Yisimayi, Yinghui Zheng, Longzhi Tan, Dong Xing, X Sunney Xie.
      Recent advances in single-cell assay for transposase accessible chromatin using sequencing (scATAC-seq) and its coassays have transformed the field of single-cell epigenomics and transcriptomics. However, the low detection efficiency of current methods has limited our understanding of the true complexity of chromatin accessibility and its relationship with gene expression in single cells. Here, we report a high-sensitivity scATAC-seq method, termed multiplexed end-tagging amplification of transposase accessible chromatin (METATAC), which detects a large number of accessible sites per cell and is compatible with automation. Our high detectability and statistical framework allowed precise linking of enhancers to promoters without merging single cells. We systematically investigated allele-specific accessibility in the mouse cerebral cortex, revealing allele-specific accessibility of promotors of certain imprinted genes but biallelic accessibility of their enhancers. Finally, we combined METATAC with our high-sensitivity single-cell RNA sequencing (scRNA-seq) method, multiple annealing and looping based amplification cycles for digital transcriptomics (MALBAC-DT), to develop a joint ATAC-RNA assay, termed METATAC and MALBAC-DT coassay by sequencing (M2C-seq). M2C-seq achieved significant improvements for both ATAC and RNA compared with previous methods, with consistent performance across cell lines and early mouse embryos.
    Keywords:  coaccessibility; imprinted gene; joint ATAC–RNA; scATAC-seq
    DOI:  https://doi.org/10.1073/pnas.2206450119
  19. Nucleic Acids Res. 2022 Sep 26. pii: gkac816. [Epub ahead of print]
    Cell Taxonomy: a curated repository of cell types with multifaceted characterization.
    Shuai Jiang, Qiheng Qian, Tongtong Zhu, Wenting Zong, Yunfei Shang, Tong Jin, Yuansheng Zhang, Ming Chen, Zishan Wu, Yuan Chu, Rongqin Zhang, Sicheng Luo, Wei Jing, Dong Zou, Yiming Bao, Jingfa Xiao, Zhang Zhang.
      Single-cell studies have delineated cellular diversity and uncovered increasing numbers of previously uncharacterized cell types in complex tissues. Thus, synthesizing growing knowledge of cellular characteristics is critical for dissecting cellular heterogeneity, developmental processes and tumorigenesis at single-cell resolution. Here, we present Cell Taxonomy (https://ngdc.cncb.ac.cn/celltaxonomy), a comprehensive and curated repository of cell types and associated cell markers encompassing a wide range of species, tissues and conditions. Combined with literature curation and data integration, the current version of Cell Taxonomy establishes a well-structured taxonomy for 3,143 cell types and houses a comprehensive collection of 26,613 associated cell markers in 257 conditions and 387 tissues across 34 species. Based on 4,299 publications and single-cell transcriptomic profiles of ∼3.5 million cells, Cell Taxonomy features multifaceted characterization for cell types and cell markers, involving quality assessment of cell markers and cell clusters, cross-species comparison, cell composition of tissues and cellular similarity based on markers. Taken together, Cell Taxonomy represents a fundamentally useful reference to systematically and accurately characterize cell types and thus lays an important foundation for deeply understanding and exploring cellular biology in diverse species.
    DOI:  https://doi.org/10.1093/nar/gkac816
  20. ACS Synth Biol. 2022 Sep 26.
    Quantification of Genome Editing and Transcriptional Control Capabilities Reveals Hierarchies among Diverse CRISPR/Cas Systems in Human Cells.
    Mario Escobar, Jing Li, Aditi Patel, Shizhe Liu, Qi Xu, Isaac B Hilton.
      CRISPR/Cas technologies have revolutionized the ability to redesign genomic information and tailor endogenous gene expression. Nevertheless, the discovery and development of new CRISPR/Cas systems has resulted in a lack of clarity surrounding the relative efficacies among these technologies in human cells. This deficit makes the optimal selection of CRISPR/Cas technologies in human cells unnecessarily challenging, which in turn hampers their adoption, and thus ultimately limits their utility. Here, we designed a series of endogenous testbed systems to methodically quantify and compare the genome editing, CRISPRi, and CRISPRa capabilities among 10 different natural and engineered Cas protein variants spanning Type II and Type V CRISPR/Cas families. We show that although all Cas protein variants are capable of genome editing and transcriptional control in human cells, hierarchies exist, particularly for genome editing and CRISPRa applications, wherein Cas9 ≥ Cas12a > Cas12e/Cas12j. Our findings also highlight the utility of our modular testbed platforms to rapidly and systematically quantify the functionality of practically any natural or engineered genomic-targeting Cas protein in human cells.
    Keywords:  CRISPR/Cas systems; CRISPRa; CRISPRi; gene regulation; genome editing
    DOI:  https://doi.org/10.1021/acssynbio.2c00156
  21. Nucleic Acids Res. 2022 Sep 27. pii: gkac820. [Epub ahead of print]
    qPTM: an updated database for PTM dynamics in human, mouse, rat and yeast.
    Kai Yu, Ye Wang, Yongqiang Zheng, Zekun Liu, Qingfeng Zhang, Siyu Wang, Qi Zhao, Xiaolong Zhang, Xiaoxing Li, Rui-Hua Xu, Ze-Xian Liu.
      Post-translational modifications (PTMs) are critical molecular mechanisms that regulate protein functions temporally and spatially in various organisms. Since most PTMs are dynamically regulated, quantifying PTM events under different states is crucial for understanding biological processes and diseases. With the rapid development of high-throughput proteomics technologies, massive quantitative PTM proteome datasets have been generated. Thus, a comprehensive one-stop data resource for surfing big data will benefit the community. Here, we updated our previous phosphorylation dynamics database qPhos to the qPTM (http://qptm.omicsbio.info). In qPTM, 11 482 553 quantification events among six types of PTMs, including phosphorylation, acetylation, glycosylation, methylation, SUMOylation and ubiquitylation in four different organisms were collected and integrated, and the matched proteome datasets were included if available. The raw mass spectrometry based false discovery rate control and the recurrences of identifications among datasets were integrated into a scoring system to assess the reliability of the PTM sites. Browse and search functions were improved to facilitate users in swiftly and accurately acquiring specific information. The results page was revised with more abundant annotations, and time-course dynamics data were visualized in trend lines. We expected the qPTM database to be a much more powerful and comprehensive data repository for the PTM research community.
    DOI:  https://doi.org/10.1093/nar/gkac820
  22. Cell Rep Methods. 2022 Sep 19. 2(9): 100288
    Experimental reproducibility limits the correlation between mRNA and protein abundances in tumor proteomic profiles.
    Swathi Ramachandra Upadhya, Colm J Ryan.
      Large-scale studies of human proteomes have revealed only a moderate correlation between mRNA and protein abundances. It is unclear to what extent this moderate correlation reflects post-transcriptional regulation and to what extent it reflects measurement error. Here, by analyzing replicate profiles of tumors and cell lines, we show that there is considerable variation in the reproducibility of measurements of transcripts and proteins from individual genes. Proteins with more reproducible measurements tend to have a higher mRNA-protein correlation, suggesting that measurement reproducibility accounts for a substantial fraction of the unexplained variation between mRNA and protein abundances. The reproducibility of individual proteins is somewhat consistent across studies, and we exploit this to develop an aggregate reproducibility score that explains a substantial amount of the variation in mRNA-protein correlations across multiple studies. Finally, we show that pathways previously reported to have a higher-than-average mRNA-protein correlation may simply contain members that can be more reproducibly quantified.
    Keywords:  cancer; gene expression; machine learning; post-transcriptional regulation; proteogenomics; proteomics; reproducibility; transcriptomics
    DOI:  https://doi.org/10.1016/j.crmeth.2022.100288
  23. Trends Biochem Sci. 2022 Sep 23. pii: S0968-0004(22)00236-5. [Epub ahead of print]
    Determining and interpreting protein lifetimes in mammalian tissues.
    Eugenio F Fornasiero, Jeffrey N Savas.
      The orchestration of protein production and degradation, and the regulation of protein lifetimes, play a central role in the majority of biological processes. Recent advances in proteomics have enabled the estimation of protein half-lives for thousands of proteins in vivo. What is the utility of these measurements, and how can they be leveraged to interpret the proteome changes occurring during development, aging, and disease? This opinion article summarizes leading technical approaches and highlights their strengths and weaknesses. We also disambiguate frequently used terminology, illustrate recent mechanistic insights, and provide guidance for interpreting and validating protein turnover measurements. Overall, protein lifetimes, coupled to estimates of protein levels, are essential for obtaining a deep understanding of mammalian biology and the basic processes defining life itself.
    Keywords:  long-lived proteins; metabolic labeling; protein half-life; protein turnover; proteomics; stable isotopes
    DOI:  https://doi.org/10.1016/j.tibs.2022.08.011
  24. Science. 2022 Sep 30. 377(6614): 1488-1489
    Metabolite-driven antitumor immunity.
    James A Nathan.
      An oncometabolite blocks T cell killing by inhibiting glycolysis.
    DOI:  https://doi.org/10.1126/science.ade3697
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