bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2021‒11‒28
twenty-five papers selected by
Ralitsa Radostinova Madsen
University College London Cancer Institute
  1. Fluctuations in AKT and PTEN Activity Are Linked by the E3 Ubiquitin Ligase cCBL.
  2. Novel Mechanisms of Tumor Promotion by the Insulin Receptor Isoform A in Triple-Negative Breast Cancer Cells.
  3. Structural effects of morpholine replacement in ZSTK474 on Class I PI3K isoform inhibition: Development of novel MEK/PI3K bifunctional inhibitors.
  4. p300 or CBP is required for insulin-stimulated glucose uptake in skeletal muscle and adipocytes.
  5. MCMICRO: a scalable, modular image-processing pipeline for multiplexed tissue imaging.
  6. CRISPR-enhanced human adipocyte browning as cell therapy for metabolic disease.
  7. A benchmark study of simulation methods for single-cell RNA sequencing data.
  8. Retraction: Overexpression of the Insulin Receptor Isoform A Promotes Endometrial Carcinoma Cell Growth.
  9. Click chemistry-enabled CRISPR screening reveals GSK3 as a regulator of PLD signaling.
  10. As cancer grows old.
  11. Metabolic stress induces GD2+ cancer stem cell-like phenotype in triple-negative breast cancer.
  12. Comprehensive Insulin Receptor Phosphorylation Dynamics Profiled by Mass Spectrometry.
  13. The structure of the human cell cycle.
  14. Simulations of Kindlin-2 PIP Binding Domains Reveal Protonation-Dependent Membrane Binding Modes.
  15. Pharmacologically controlling protein-protein interactions through epichaperomes for therapeutic vulnerability in cancer.
  16. Genome-wide CRISPR screen identifies CDK6 as a therapeutic target in Adult T-cell leukemia/lymphoma.
  17. Genome-wide CRISPR-Cas9 screens identify mechanisms of BET bromodomain inhibitor sensitivity.
  18. Benign tumors in TSC are amenable to treatment by GD3 CAR T cells in mice.
  19. Substrate stiffening promotes VEGF-A functions via the PI3K/Akt/mTOR pathway.
  20. The amoeboid state as part of the epithelial-to-mesenchymal transition programme.
  21. Targeting p21Cip1 highly expressing cells in adipose tissue alleviates insulin resistance in obesity.
  22. Spatial mapping of protein composition and tissue organization: a primer for multiplexed antibody-based imaging.
  23. ecDNA hubs drive cooperative intermolecular oncogene expression.
  24. An atlas of protein turnover rates in mouse tissues.
  25. B-cell receptor isotypes differentially associate with cell signaling, kinetics, and outcome in chronic lymphocytic leukemia.
  1. Cells. 2021 Oct 20. pii: 2803. [Epub ahead of print]10(11):
    Fluctuations in AKT and PTEN Activity Are Linked by the E3 Ubiquitin Ligase cCBL.
    Manuel Olazábal-Morán, Miriam Sánchez-Ortega, Laura Martínez-Muñoz, Carmen Hernández, Manuel S Rodríguez, Mario Mellado, Ana C Carrera.
      3-Poly-phosphoinositides (PIP3) regulate cell survival, division, and migration. Both PI3-kinase (phosphoinositide-3-kinase) and PTEN (phosphatase and tensin-homolog in chromosome 10) control PIP3 levels, but the mechanisms connecting PI3-kinase and PTEN are unknown. Using non-transformed cells, the activation kinetics of PTEN and of the PIP3-effector AKT were examined after the addition of growth factors. Both epidermal growth factor and serum induced the early activation of AKT and the simultaneous inactivation of PTEN (at ~5 min). This PIP3/AKT peak was followed by a general reduction in AKT activity coincident with the recovery of PTEN phosphatase activity (at ~10-15 min). Subsequent AKT peaks and troughs followed. The fluctuation in AKT activity was linked to that of PTEN; PTEN reconstitution in PTEN-null cells restored AKT fluctuations, while PTEN depletion in control cells abrogated them. The analysis of PTEN activity fluctuations after the addition of growth factors showed its inactivation at ~5 min to be simultaneous with its transient ubiquitination, which was regulated by the ubiquitin E3 ligase cCBL (casitas B-lineage lymphoma proto-oncogene). Protein-protein interaction analysis revealed cCBL to be brought into the proximity of PTEN in a PI3-kinase-dependent manner. These results reveal a mechanism for PI3-kinase/PTEN crosstalk and suggest that cCBL could be new target in strategies designed to modulate PTEN activity in cancer.
    Keywords:  AKT; CBL; E3 ubiquitin ligase; PTEN; phosphatidylinositide 3-kinase
    DOI:  https://doi.org/10.3390/cells10112803
  2. Cells. 2021 Nov 12. pii: 3145. [Epub ahead of print]10(11):
    Novel Mechanisms of Tumor Promotion by the Insulin Receptor Isoform A in Triple-Negative Breast Cancer Cells.
    Veronica Vella, Marika Giuliano, Alessandro La Ferlita, Michele Pellegrino, Germano Gaudenzi, Salvatore Alaimo, Michele Massimino, Alfredo Pulvirenti, Alessandra Dicitore, Paolo Vigneri, Giovanni Vitale, Roberta Malaguarnera, Andrea Morrione, Andrew H Sims, Alfredo Ferro, Marcello Maggiolini, Rosamaria Lappano, Ernestina Marianna De Francesco, Antonino Belfiore.
      The insulin receptor isoform A (IR-A) plays an increasingly recognized role in fetal growth and tumor biology in response to circulating insulin and/or locally produced IGF2. This role seems not to be shared by the IR isoform B (IR-B). We aimed to dissect the specific impact of IR isoforms in modulating insulin signaling in triple negative breast cancer (TNBC) cells. We generated murine 4T1 TNBC cells deleted from the endogenous insulin receptor (INSR) gene and expressing comparable levels of either human IR-A or IR-B. We then measured IR isoform-specific in vitro and in vivo biological effects and transcriptome in response to insulin. Overall, the IR-A was more potent than the IR-B in mediating cell migration, invasion, and in vivo tumor growth. Transcriptome analysis showed that approximately 89% of insulin-stimulated transcripts depended solely on the expression of the specific isoform. Notably, in cells overexpressing IR-A, insulin strongly induced genes involved in tumor progression and immune evasion including chemokines and genes related to innate immunity. Conversely, in IR-B overexpressing cells, insulin predominantly induced the expression of genes primarily involved in the regulation of metabolic pathways and, to a lesser extent, tumor growth and angiogenesis.
    Keywords:  IGF axis; breast cancer; hyperinsulinemia; insulin receptor isoform A; insulin receptor isoform transcriptome; insulin receptor isoforms; triple negative breast cancer; tumor promotion
    DOI:  https://doi.org/10.3390/cells10113145
  3. Eur J Med Chem. 2021 Nov 14. pii: S0223-5234(21)00845-X. [Epub ahead of print] 113996
    Structural effects of morpholine replacement in ZSTK474 on Class I PI3K isoform inhibition: Development of novel MEK/PI3K bifunctional inhibitors.
    Marcian E Van Dort, Yongsoon Jang, Christopher A Bonham, Kevin Heist, Dilrukshika S W Palagama, Lucas McDonald, Edward Z Zhang, Thomas L Chenevert, Gary D Luker, Brian D Ross.
      Established roles for PI3K and MAPK signaling pathways in tumorigenesis has prompted extensive research towards the discovery of small-molecule inhibitors as cancer therapeutics. However, significant compensatory regulation exists between these two signaling cascades, leading to redundancy among survival pathways. Consequently, initial clinical trials aimed at either PI3K or MEK inhibition alone have proven ineffective and highlight the need for development of targeted and innovative therapeutic combination strategies. We designed a series of PI3K inhibitor derivatives wherein a single morpholine group of the PI3K inhibitor ZSTK474 was substituted with a variety of 2-aminoethyl functional groups. Analogs with pendant hydroxyl or methoxy groups maintained low nanomolar inhibition towards PI3Kα, PI3Kγ, and PI3Kδ isoforms in contrast to those with pendant amino groups which were significantly less inhibitory. Synthesis of prototype PI3K/MEK bifunctional inhibitors (6r, 6s) was guided by the structure-activity data, where a MEK-targeting inhibitor was tethered directly via a short PEG linker to the triazine core of the PI3K inhibitor analogs. These compounds (6r, 6s) displayed nanomolar inhibition towards PI3Kα, δ, and MEK (IC50 ∼105-350 nM), and low micromolar inhibition for PI3Kβ and PI3Kγ (IC50 ∼1.5-3.9 μM) in enzymatic inhibition assays. Cell viability assays demonstrated superior anti-proliferative activity for 6s over 6r in three tumor-derived cell lines (A375, D54, SET-2), which correlated with inhibition of downstream AKT and ERK1/2 phosphorylation. Compounds 6r and 6s also demonstrated in vivo tolerability with therapeutic efficacy through reduction of kinase activation and amelioration of disease phenotypes in the JAK2V617F mutant myelofibrosis mouse cancer model. Taken together, these results support further structure optimization of 6r and 6s as promising leads for combination therapy in human cancer as a new class of PI3K/MEK bifunctional inhibitors.
    Keywords:  Bifunctional MEK/PI3K inhibitor; PI3K isoform inhibition; ZSTK474
    DOI:  https://doi.org/10.1016/j.ejmech.2021.113996
  4. JCI Insight. 2021 Nov 23. pii: e141344. [Epub ahead of print]
    p300 or CBP is required for insulin-stimulated glucose uptake in skeletal muscle and adipocytes.
    Vitor F Martins, Samuel A LaBarge, Alexandra Stanley, Kristoffer Svensson, Chao-Wei Hung, Omer Keinan, Theodore P Ciaraldi, Dion Banoian, Ji E Park, Christina Ha, Byron Hetrick, Gretchen A Meyer, Andrew Philp, Larry L David, Robert R Henry, Joseph E Aslan, Alan R Saltiel, Carrie E McCurdy, Simon Schenk.
      While current thinking posits that insulin signaling to GLUT4 exocytic translocation and glucose uptake in skeletal muscle and adipocytes is controlled by phosphorylation-based signaling, many proteins in this pathway are acetylated on lysine residues. However, the importance of acetylation and lysine acetyltransferases to insulin-stimulated glucose uptake is incompletely defined. Here, we demonstrate that combined loss of the acetyltransferases E1A binding protein p300 (p300) and cAMP response element binding protein binding protein (CBP) in mouse skeletal muscle causes a complete loss of insulin-stimulated glucose uptake. Similarly, brief (i.e. 1 h) pharmacological inhibition of p300/CBP acetyltransferase activity recapitulates this phenotype in human and rodent myotubes, 3T3-L1 adipocytes, and mouse muscle. Mechanistically, these effects are due to p300/CBP-mediated regulation of GLUT4 exocytic translocation and occurs downstream of Akt signaling. Taken together, we highlight a fundamental role for acetylation and p300/CBP in the direct regulation of insulin-stimulated glucose transport in skeletal muscle and adipocytes.
    Keywords:  Endocrinology; Glucose metabolism; Insulin signaling; Muscle Biology; Skeletal muscle
    DOI:  https://doi.org/10.1172/jci.insight.141344
  5. Nat Methods. 2021 Nov 25.
    MCMICRO: a scalable, modular image-processing pipeline for multiplexed tissue imaging.
    Denis Schapiro, Artem Sokolov, Clarence Yapp, Yu-An Chen, Jeremy L Muhlich, Joshua Hess, Allison L Creason, Ajit J Nirmal, Gregory J Baker, Maulik K Nariya, Jia-Ren Lin, Zoltan Maliga, Connor A Jacobson, Matthew W Hodgman, Juha Ruokonen, Samouil L Farhi, Domenic Abbondanza, Eliot T McKinley, Daniel Persson, Courtney Betts, Shamilene Sivagnanam, Aviv Regev, Jeremy Goecks, Robert J Coffey, Lisa M Coussens, Sandro Santagata, Peter K Sorger.
      Highly multiplexed tissue imaging makes detailed molecular analysis of single cells possible in a preserved spatial context. However, reproducible analysis of large multichannel images poses a substantial computational challenge. Here, we describe a modular and open-source computational pipeline, MCMICRO, for performing the sequential steps needed to transform whole-slide images into single-cell data. We demonstrate the use of MCMICRO on tissue and tumor images acquired using multiple imaging platforms, thereby providing a solid foundation for the continued development of tissue imaging software.
    DOI:  https://doi.org/10.1038/s41592-021-01308-y
  6. Nat Commun. 2021 Nov 26. 12(1): 6931
    CRISPR-enhanced human adipocyte browning as cell therapy for metabolic disease.
    Emmanouela Tsagkaraki, Sarah M Nicoloro, Tiffany DeSouza, Javier Solivan-Rivera, Anand Desai, Lawrence M Lifshitz, Yuefei Shen, Mark Kelly, Adilson Guilherme, Felipe Henriques, Nadia Amrani, Raed Ibraheim, Tomas C Rodriguez, Kevin Luk, Stacy Maitland, Randall H Friedline, Lauren Tauer, Xiaodi Hu, Jason K Kim, Scot A Wolfe, Erik J Sontheimer, Silvia Corvera, Michael P Czech.
      Obesity and type 2 diabetes are associated with disturbances in insulin-regulated glucose and lipid fluxes and severe comorbidities including cardiovascular disease and steatohepatitis. Whole body metabolism is regulated by lipid-storing white adipocytes as well as "brown" and "brite/beige" adipocytes that express thermogenic uncoupling protein 1 (UCP1) and secrete factors favorable to metabolic health. Implantation of brown fat into obese mice improves glucose tolerance, but translation to humans has been stymied by low abundance of primary human beige adipocytes. Here we apply methods to greatly expand human adipocyte progenitors from small samples of human subcutaneous adipose tissue and then disrupt the thermogenic suppressor gene NRIP1 by CRISPR. Ribonucleoprotein consisting of Cas9 and sgRNA delivered ex vivo are fully degraded by the human cells following high efficiency NRIP1 depletion without detectable off-target editing. Implantation of such CRISPR-enhanced human or mouse brown-like adipocytes into high fat diet fed mice decreases adiposity and liver triglycerides while enhancing glucose tolerance compared to implantation with unmodified adipocytes. These findings advance a therapeutic strategy to improve metabolic homeostasis through CRISPR-based genetic enhancement of human adipocytes without exposing the recipient to immunogenic Cas9 or delivery vectors.
    DOI:  https://doi.org/10.1038/s41467-021-27190-y
  7. Nat Commun. 2021 Nov 25. 12(1): 6911
    A benchmark study of simulation methods for single-cell RNA sequencing data.
    Yue Cao, Pengyi Yang, Jean Yee Hwa Yang.
      Single-cell RNA-seq (scRNA-seq) data simulation is critical for evaluating computational methods for analysing scRNA-seq data especially when ground truth is experimentally unattainable. The reliability of evaluation depends on the ability of simulation methods to capture properties of experimental data. However, while many scRNA-seq data simulation methods have been proposed, a systematic evaluation of these methods is lacking. We develop a comprehensive evaluation framework, SimBench, including a kernel density estimation measure to benchmark 12 simulation methods through 35 scRNA-seq experimental datasets. We evaluate the simulation methods on a panel of data properties, ability to maintain biological signals, scalability and applicability. Our benchmark uncovers performance differences among the methods and highlights the varying difficulties in simulating data characteristics. Furthermore, we identify several limitations including maintaining heterogeneity of distribution. These results, together with the framework and datasets made publicly available as R packages, will guide simulation methods selection and their future development.
    DOI:  https://doi.org/10.1038/s41467-021-27130-w
  8. PLoS One. 2021 ;16(11): e0260686
    Retraction: Overexpression of the Insulin Receptor Isoform A Promotes Endometrial Carcinoma Cell Growth.
    PLOS ONE Editors
      
    DOI:  https://doi.org/10.1371/journal.pone.0260686
  9. Proc Natl Acad Sci U S A. 2021 Nov 30. pii: e2025265118. [Epub ahead of print]118(48):
    Click chemistry-enabled CRISPR screening reveals GSK3 as a regulator of PLD signaling.
    Timothy W Bumpus, Shiying Huang, Reika Tei, Jeremy M Baskin.
      Enzymes that produce second messengers are highly regulated. Revealing the mechanisms underlying such regulation is critical to understanding both how cells achieve specific signaling outcomes and return to homeostasis following a particular stimulus. Pooled genome-wide CRISPR screens are powerful unbiased approaches to elucidate regulatory networks, their principal limitation being the choice of phenotype selection. Here, we merge advances in bioorthogonal fluorescent labeling and CRISPR screening technologies to discover regulators of phospholipase D (PLD) signaling, which generates the potent lipid second messenger phosphatidic acid. Our results reveal glycogen synthase kinase 3 as a positive regulator of protein kinase C and PLD signaling. More generally, this work demonstrates how bioorthogonal, activity-based fluorescent tagging can expand the power of CRISPR screening to uncover mechanisms regulating specific enzyme-driven signaling pathways in mammalian cells.
    Keywords:  CRISPR screening; CRISPRi; GSK3; click chemistry; phospholipase D
    DOI:  https://doi.org/10.1073/pnas.2025265118
  10. Science. 2021 Nov 26. 374(6571): 1066
    As cancer grows old.
    Adrian Baez-Ortega.
      [Figure: see text].
    DOI:  https://doi.org/10.1126/science.abm8137
  11. Br J Cancer. 2021 Nov 22.
    Metabolic stress induces GD2+ cancer stem cell-like phenotype in triple-negative breast cancer.
    Appalaraju Jaggupilli, Stanley Ly, Khoa Nguyen, Vivek Anand, Bin Yuan, Fouad El-Dana, Yuanqing Yan, Zoe Arvanitis, Danthasinghe Waduge Badrajee Piyarathna, Nagireddy Putluri, Helen Piwnica-Worms, Henry Charles Manning, Michael Andreeff, V Lokesh Battula.
      BACKGROUND: Metabolic stress resulting from nutrient deficiency is one of the hallmarks of a growing tumour. Here, we tested the hypothesis that metabolic stress induces breast cancer stem-like cell (BCSC) phenotype in triple-negative breast cancer (TNBC).METHODS: Flow cytometry for GD2 expression, mass spectrometry and Ingenuity Pathway Analysis for metabolomics, bioinformatics, in vitro tumorigenesis and in vivo models were used.
    RESULTS: Serum/glucose deprivation not only increased stress markers but also enhanced GD2+ BCSC phenotype and function in TNBC cells. Global metabolomics profiling identified upregulation of glutathione biosynthesis in GD2high cells, suggesting a role of glutamine in the BCSC phenotype. Cueing from the upregulation of the glutamine transporters in primary breast tumours, inhibition of glutamine uptake using small-molecule inhibitor V9302 reduced GD2+ cells by 70-80% and BCSC characteristics in TNBC cells. Mechanistic studies revealed inhibition of the mTOR pathway and induction of ferroptosis by V9302 in TNBC cells. Finally, inhibition of glutamine uptake significantly reduced in vivo tumour growth in a TNBC patient-derived xenograft model using NSG (non-obese diabetic/severe combined immunodeficiency with a complete null allele of the IL-2 receptor common gamma chain) mice.
    CONCLUSION: Here, we show metabolic stress results in GD2+ BCSC phenotype in TNBC and glutamine contributes to GD2+ phenotype, and targeting the glutamine transporters could complement conventional chemotherapy in TNBC.
    DOI:  https://doi.org/10.1038/s41416-021-01636-y
  12. FEBS J. 2021 Nov 26.
    Comprehensive Insulin Receptor Phosphorylation Dynamics Profiled by Mass Spectrometry.
    Zhongping Liao, Chen Zhang, Liyun Ding, Julie S Moyers, Jason X Tang, John M Beals.
      Insulin receptor (IR) phosphorylation is critical for the assessment of the extent of IR agonism and nuances in downstream signaling cascade. Thorough identification and monitoring of the phosphorylation events is important to understand the process of insulin signaling transduction and regulation. Although IR phosphorylation has been extensively studied in the past decades, only a handful of phosphorylation sites can be identified by either traditional antibody-based assays or recent large-scale mass spectrometry-based phosphoproteomics approaches. In this study, the most exhaustive assessment of the IR phosphorylation was conducted using nano-LC MS/MS; wherein, a total of 13 IR phosphorylation sites and 22 combinations thereof were analyzed. The kinetic analysis included Y965, Y972, S968/969, and S974/976 in the juxtamembrane region; Y1158, Y1162 and Y1163 in the kinase domain; and Y1328, Y1334, S1278, S1320, S1321, and T1348 in the C-terminal region. Employing two different receptor agonists, i.e., insulin and an IR peptide agonist, the data revealed contrasting phosphorylation kinetics across these sites with dynamics far more diverse than expected for known IR agonists. Notably, cell trafficking experiments revealed that the IR peptide agonist was incapable of inducing IR to the early endosome, which is likely linked to difference in IR phosphorylation. This work provides a powerful tool for studying IR signaling and trafficking that can benefit the design of IR agonists with improved therapeutic utility.
    Keywords:  Insulin receptor phosphorylation; insulin receptor endocytosis; mass spectrometry
    DOI:  https://doi.org/10.1111/febs.16299
  13. Cell Syst. 2021 Nov 17. pii: S2405-4712(21)00418-X. [Epub ahead of print]
    The structure of the human cell cycle.
    Wayne Stallaert, Katarzyna M Kedziora, Colin D Taylor, Tarek M Zikry, Jolene S Ranek, Holly K Sobon, Sovanny R Taylor, Catherine L Young, Jeanette G Cook, Jeremy E Purvis.
      Understanding the organization of the cell cycle has been a longstanding goal in cell biology. We combined time-lapse microscopy, highly multiplexed single-cell imaging of 48 core cell cycle proteins, and manifold learning to render a visualization of the human cell cycle. This data-driven approach revealed the comprehensive "structure" of the cell cycle: a continuum of molecular states that cells occupy as they transition from one cell division to the next, or as they enter or exit cell cycle arrest. Paradoxically, progression deeper into cell cycle arrest was accompanied by increases in proliferative effectors such as CDKs and cyclins, which can drive cell cycle re-entry by overcoming p21 induction. The structure also revealed the molecular trajectories into senescence and the unique combination of molecular features that define this irreversibly arrested state. This approach will enable the comparison of alternative cell cycles during development, in response to environmental perturbation and in disease. A record of this paper's transparent peer review process is included in the supplemental information.
    Keywords:  cell cycle; machine learning; manifold learning; quiescence; senescence; single-cell imaging
    DOI:  https://doi.org/10.1016/j.cels.2021.10.007
  14. Biophys J. 2021 Nov 20. pii: S0006-3495(21)00990-5. [Epub ahead of print]
    Simulations of Kindlin-2 PIP Binding Domains Reveal Protonation-Dependent Membrane Binding Modes.
    Robert D Palmere, David A Case, Andrew J Nieuwkoop.
      Kindlin-2, a member of the Kindlin family of peripheral membrane proteins, is important for integrin activation and stabilization of epidermal growth factor receptor. It associates with the cytoplasmic face of the plasma membrane via dedicated phosphatidylinositol phosphate binding domains located in the N-terminal F0 and Pleckstrin Homology domains. These domains have binding affinity for phosphatidylinositol 4,5-bisphosphate and, to a greater degree phosphatidylinositol 3,4,5-trisphosphate. The biological significance of the differential binding of these phosphatidylinositol phosphates to Kindlin-2 and the mechanism by which they activate Kindlin-2 are not well understood. Recently, ssNMR identified the predominant protonation states of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate near physiological pH in the presence of anionic lipids. Here, we perform atomistic simulation of the bound state of the Pleckstrin Homology and F0 domains of Kindlin-2 at membranes containing phosphatidylinositol 4,5-bisphosphate / phosphatidylinositol 3,4,5-trisphosphate with differing protonation states. This computational approach demonstrates that these two phosphatidylinositol phosphates differently modulate Kindlin-2 subdomain binding in a protonation state dependent manner. We speculate these variations in binding mode provide a mechanism for intracellular pH and Ca2+ influx to control the membrane binding behavior and activity of Kindlin-2.
    Keywords:  Kindlin-2; Mig-2; inositol; lipid bilayer; membrane; phosphatidylinositol (3,4,5)-trisphosphate; phosphatidylinositol (4,5)-bisphosphate
    DOI:  https://doi.org/10.1016/j.bpj.2021.11.021
  15. Commun Biol. 2021 Nov 25. 4(1): 1333
    Pharmacologically controlling protein-protein interactions through epichaperomes for therapeutic vulnerability in cancer.
    Suhasini Joshi, Erica DaGama Gomes, Tai Wang, Adriana Corben, Tony Taldone, Srinivasa Gandu, Chao Xu, Sahil Sharma, Salma Buddaseth, Pengrong Yan, Lon Yin L Chan, Askan Gokce, Vinagolu K Rajasekhar, Lisa Shrestha, Palak Panchal, Justina Almodovar, Chander S Digwal, Anna Rodina, Swathi Merugu, NagaVaraKishore Pillarsetty, Vlad Miclea, Radu I Peter, Wanyan Wang, Stephen D Ginsberg, Laura Tang, Marissa Mattar, Elisa de Stanchina, Kenneth H Yu, Maeve Lowery, Olivera Grbovic-Huezo, Eileen M O'Reilly, Yelena Janjigian, John H Healey, William R Jarnagin, Peter J Allen, Chris Sander, Hediye Erdjument-Bromage, Thomas A Neubert, Steven D Leach, Gabriela Chiosis.
      Cancer cell plasticity due to the dynamic architecture of interactome networks provides a vexing outlet for therapy evasion. Here, through chemical biology approaches for systems level exploration of protein connectivity changes applied to pancreatic cancer cell lines, patient biospecimens, and cell- and patient-derived xenografts in mice, we demonstrate interactomes can be re-engineered for vulnerability. By manipulating epichaperomes pharmacologically, we control and anticipate how thousands of proteins interact in real-time within tumours. Further, we can essentially force tumours into interactome hyperconnectivity and maximal protein-protein interaction capacity, a state whereby no rebound pathways can be deployed and where alternative signalling is supressed. This approach therefore primes interactomes to enhance vulnerability and improve treatment efficacy, enabling therapeutics with traditionally poor performance to become highly efficacious. These findings provide proof-of-principle for a paradigm to overcome drug resistance through pharmacologic manipulation of proteome-wide protein-protein interaction networks.
    DOI:  https://doi.org/10.1038/s42003-021-02842-3
  16. Blood. 2021 Nov 24. pii: blood.2021012734. [Epub ahead of print]
    Genome-wide CRISPR screen identifies CDK6 as a therapeutic target in Adult T-cell leukemia/lymphoma.
    Takashi Ishio, Sarvesh Kumar, Joji Shimono, Anusara Daenthanasanmak, Sigrid Dubois, Yuquan Lin, Bonita R Bryant, Michael N Petrus, Emmanuel Bachy, Da Wei Huang, Yandan Yang, Patrick L Green, Hiroo Hasegawa, Michiyuki Maeda, Hideki Goto, Tomoyuki Endo, Takashi Yokota, Kanako C Hatanaka, Yutaka Hatanaka, Shinya Tanaka, Yoshihiro Matsuno, Yibin Yang, Satoshi Hashino, Takanori Teshima, Thomas A Waldmann, Louis M Staudt, Masao Nakagawa.
      Adult T-cell leukemia/lymphoma (ATLL) is an aggressive T-cell malignancy with a poor prognosis with current therapy. Here we report genome-wide CRISPR-Cas9 screening of ATLL models, which identified CDK6, CCND2, BATF3, JUNB, STAT3, and IL10RB as genes that are essential for the proliferation and/or survival of ATLL cells. As a single agent, the CDK6 inhibitor palbociclib induced cell cycle arrest and apoptosis in ATLL models with wild type TP53. ATLL models that had inactivated TP53 genetically were relatively resistant to palbociclib owing to compensatory CDK2 activity, and this resistance could be reversed by APR-246, a small molecule activator of mutant TP53. The CRISPR-Cas9 screen further highlighted the dependence of ATLL cells on mTORC1 signaling. Treatment of ATLL cells with palbociclib in combination with mTORC1 inhibitors was synergistically toxic irrespective of the TP53 status. This work defines CDK6 as a novel therapeutic target for ATLL and supports the clinical evaluation of palbociclib in combination with mTORC1 inhibitors in this recalcitrant malignancy.
    DOI:  https://doi.org/10.1182/blood.2021012734
  17. iScience. 2021 Nov 19. 24(11): 103323
    Genome-wide CRISPR-Cas9 screens identify mechanisms of BET bromodomain inhibitor sensitivity.
    David Estoppey, Gabi Schutzius, Christian Kolter, Adrian Salathe, Tiffany Wunderlin, Amandine Meyer, Florian Nigsch, Tewis Bouwmeester, Dominic Hoepfner, Susan Kirkland.
      BET bromodomain inhibitors hold promise as therapeutic agents in diverse indications, but their clinical progression has been challenging and none have received regulatory approval. Early clinical trials in cancer have shown heterogeneous clinical responses, development of resistance, and adverse events. Increased understanding of their mechanism(s) of action and identification of biomarkers are needed to identify appropriate indication(s) and achieve efficacious dosing. Using genome-wide CRISPR-Cas9 screens at different concentrations, we report molecular mechanisms defining cellular responses to BET inhibitors, some of which appear specific to a single compound concentration. We identify multiple transcriptional regulators and mTOR pathway members as key determinants of JQ1 sensitivity and two Ca2+/Mn2+ transporters, ATP2C1 and TMEM165, as key determinants of JQ1 resistance. Our study reveals new molecular mediators of BET bromodomain inhibitor effects, suggests the involvement of manganese, and provides a rich resource for discovery of biomarkers and targets for combination therapies.
    Keywords:  Cell biology; Chemogenomics; Molecular biology
    DOI:  https://doi.org/10.1016/j.isci.2021.103323
  18. JCI Insight. 2021 Nov 22. pii: e152014. [Epub ahead of print]6(22):
    Benign tumors in TSC are amenable to treatment by GD3 CAR T cells in mice.
    Ancy Thomas, Saurav Sumughan, Emilia R Dellacecca, Rohan S Shivde, Nicola Lancki, Zhussipbek Mukhatayev, Cristina C Vaca, Fei Han, Levi Barse, Steven W Henning, Jesus Zamora-Pineda, Suhail Akhtar, Nikhilesh Gupta, Jasmine O Zahid, Stephanie R Zack, Prathyaya Ramesh, Dinesh Jaishankar, Agnes Sy Lo, Joel Moss, Maria M Picken, Thomas N Darling, Denise M Scholtens, Daniel F Dilling, Richard P Junghans, I Caroline Le Poole.
      Mutations underlying disease in tuberous sclerosis complex (TSC) give rise to tumors with biallelic mutations in TSC1 or TSC2 and hyperactive mammalian target of rapamycin complex 1 (mTORC1). Benign tumors might exhibit de novo expression of immunogens, targetable by immunotherapy. As tumors may rely on ganglioside D3 (GD3) expression for mTORC1 activation and growth, we compared GD3 expression in tissues from patients with TSC and controls. GD3 was overexpressed in affected tissues from patients with TSC and also in aging Tsc2+/- mice. As GD3 overexpression was not accompanied by marked natural immune responses to the target molecule, we performed preclinical studies with GD3 chimeric antigen receptor (CAR) T cells. Polyfunctional CAR T cells were cytotoxic toward GD3-overexpressing targets. In mice challenged with Tsc2-/- tumor cells, CAR T cells substantially and durably reduced the tumor burden, correlating with increased T cell infiltration. We also treated aged Tsc2+/- heterozygous (>60 weeks) mice that carry spontaneous Tsc2-/- tumors with GD3 CAR or untransduced T cells and evaluated them at endpoint. Following CAR T cell treatment, the majority of mice were tumor free while all control animals carried tumors. The outcomes demonstrate a strong treatment effect and suggest that targeting GD3 can be successful in TSC.
    Keywords:  Antigen; Immunology; Immunotherapy; T cells; Therapeutics
    DOI:  https://doi.org/10.1172/jci.insight.152014
  19. Biochem Biophys Res Commun. 2021 Nov 13. pii: S0006-291X(21)01541-2. [Epub ahead of print]586 27-33
    Substrate stiffening promotes VEGF-A functions via the PI3K/Akt/mTOR pathway.
    Amjad Husain, Arogya Khadka, Allen Ehrlicher, Magali Saint-Geniez, Ramaswamy Krishnan.
      While it is now well-established that substrate stiffness regulates vascular endothelial growth factor-A (VEGF-A) mediated signaling and functions, causal mechanisms remain poorly understood. Here, we report an underlying role for the PI3K/Akt/mTOR signaling pathway. This pathway is activated on stiffer substrates, is amplified by VEGF-A stimulation, and correlates with enhanced endothelial cell (EC) proliferation, contraction, pro-angiogenic secretion, and capillary-like tube formation. In the settings of advanced age-related macular degeneration, characterized by EC and retinal pigment epithelial (RPE)-mediated angiogenesis, these data implicate substrate stiffness as a novel causative mechanism and Akt/mTOR inhibition as a novel therapeutic pathway.
    Keywords:  AMD; Angiogenesis; Contraction; Endothelial cell; Proliferation; RPE; Rigidity
    DOI:  https://doi.org/10.1016/j.bbrc.2021.11.030
  20. Trends Cell Biol. 2021 Nov 23. pii: S0962-8924(21)00206-3. [Epub ahead of print]
    The amoeboid state as part of the epithelial-to-mesenchymal transition programme.
    Vittoria Graziani, Irene Rodriguez-Hernandez, Oscar Maiques, Victoria Sanz-Moreno.
      Cell migration is essential for many biological processes, while abnormal cell migration is characteristic of cancer cells. Epithelial cells become motile by undergoing epithelial-to-mesenchymal transition (EMT), and mesenchymal cells increase migration speed by adopting amoeboid features. This review highlights how amoeboid behaviour is not merely a migration mode but rather a cellular state - within the EMT spectra - by which cancer cells survive, invade and colonise challenging microenvironments. Molecular biomarkers and physicochemical triggers associated with amoeboid behaviour are discussed, including an amoeboid associated tumour microenvironment. We reflect on how amoeboid characteristics support metastasis and how their liabilities could turn into therapeutic opportunities.
    Keywords:  actomyosin; amoeboid migration; cancer stem cells; epithelial-to-mesenchymal transition; immunosuppression; therapy resistance
    DOI:  https://doi.org/10.1016/j.tcb.2021.10.004
  21. Cell Metab. 2021 Nov 19. pii: S1550-4131(21)00530-1. [Epub ahead of print]
    Targeting p21Cip1 highly expressing cells in adipose tissue alleviates insulin resistance in obesity.
    Lichao Wang, Binsheng Wang, Nathan S Gasek, Yueying Zhou, Rachel L Cohn, Dominique E Martin, Wulin Zuo, William F Flynn, Chun Guo, Evan R Jellison, Taewan Kim, Larissa G P Langhi Prata, Allyson K Palmer, Ming Li, Christina L Inman, Lauren S Barber, Iman M A Al-Naggar, Yanjiao Zhou, George A Kuchel, Alexander Meves, Tamar Tchkonia, James L Kirkland, Paul Robson, Ming Xu.
      Insulin resistance is a pathological state often associated with obesity, representing a major risk factor for type 2 diabetes. Limited mechanism-based strategies exist to alleviate insulin resistance. Here, using single-cell transcriptomics, we identify a small, critically important, but previously unexamined cell population, p21Cip1 highly expressing (p21high) cells, which accumulate in adipose tissue with obesity. By leveraging a p21-Cre mouse model, we demonstrate that intermittent clearance of p21high cells can both prevent and alleviate insulin resistance in obese mice. Exclusive inactivation of the NF-κB pathway within p21high cells, without killing them, attenuates insulin resistance. Moreover, fat transplantation experiments establish that p21high cells within fat are sufficient to cause insulin resistance in vivo. Importantly, a senolytic cocktail, dasatinib plus quercetin, eliminates p21high cells in human fat ex vivo and mitigates insulin resistance following xenotransplantation into immuno-deficient mice. Our findings lay the foundation for pursuing the targeting of p21high cells as a new therapy to alleviate insulin resistance.
    Keywords:  Cellular senescence; NF-κB; diabetes; fat transplantation; senolytics; xenograft
    DOI:  https://doi.org/10.1016/j.cmet.2021.11.002
  22. Nat Methods. 2021 Nov 22.
    Spatial mapping of protein composition and tissue organization: a primer for multiplexed antibody-based imaging.
    John W Hickey, Elizabeth K Neumann, Andrea J Radtke, Jeannie M Camarillo, Rebecca T Beuschel, Alexandre Albanese, Elizabeth McDonough, Julia Hatler, Anne E Wiblin, Jeremy Fisher, Josh Croteau, Eliza C Small, Anup Sood, Richard M Caprioli, R Michael Angelo, Garry P Nolan, Kwanghun Chung, Stephen M Hewitt, Ronald N Germain, Jeffrey M Spraggins, Emma Lundberg, Michael P Snyder, Neil L Kelleher, Sinem K Saka.
      Tissues and organs are composed of distinct cell types that must operate in concert to perform physiological functions. Efforts to create high-dimensional biomarker catalogs of these cells have been largely based on single-cell sequencing approaches, which lack the spatial context required to understand critical cellular communication and correlated structural organization. To probe in situ biology with sufficient depth, several multiplexed protein imaging methods have been recently developed. Though these technologies differ in strategy and mode of immunolabeling and detection tags, they commonly utilize antibodies directed against protein biomarkers to provide detailed spatial and functional maps of complex tissues. As these promising antibody-based multiplexing approaches become more widely adopted, new frameworks and considerations are critical for training future users, generating molecular tools, validating antibody panels, and harmonizing datasets. In this Perspective, we provide essential resources, key considerations for obtaining robust and reproducible imaging data, and specialized knowledge from domain experts and technology developers.
    DOI:  https://doi.org/10.1038/s41592-021-01316-y
  23. Nature. 2021 Nov 24.
    ecDNA hubs drive cooperative intermolecular oncogene expression.
    King L Hung, Kathryn E Yost, Liangqi Xie, Quanming Shi, Konstantin Helmsauer, Jens Luebeck, Robert Schöpflin, Joshua T Lange, Rocío Chamorro González, Natasha E Weiser, Celine Chen, Maria E Valieva, Ivy Tsz-Lo Wong, Sihan Wu, Siavash R Dehkordi, Connor V Duffy, Katerina Kraft, Jun Tang, Julia A Belk, John C Rose, M Ryan Corces, Jeffrey M Granja, Rui Li, Utkrisht Rajkumar, Jordan Friedlein, Anindya Bagchi, Ansuman T Satpathy, Robert Tjian, Stefan Mundlos, Vineet Bafna, Anton G Henssen, Paul S Mischel, Zhe Liu, Howard Y Chang.
      Extrachromosomal DNA (ecDNA) is prevalent in human cancers and mediates high expression of oncogenes through gene amplification and altered gene regulation1. Gene induction typically involves cis-regulatory elements that contact and activate genes on the same chromosome2,3. Here we show that ecDNA hubs-clusters of around 10-100 ecDNAs within the nucleus-enable intermolecular enhancer-gene interactions to promote oncogene overexpression. ecDNAs that encode multiple distinct oncogenes form hubs in diverse cancer cell types and primary tumours. Each ecDNA is more likely to transcribe the oncogene when spatially clustered with additional ecDNAs. ecDNA hubs are tethered by the bromodomain and extraterminal domain (BET) protein BRD4 in a MYC-amplified colorectal cancer cell line. The BET inhibitor JQ1 disperses ecDNA hubs and preferentially inhibits ecDNA-derived-oncogene transcription. The BRD4-bound PVT1 promoter is ectopically fused to MYC and duplicated in ecDNA, receiving promiscuous enhancer input to drive potent expression of MYC. Furthermore, the PVT1 promoter on an exogenous episome suffices to mediate gene activation in trans by ecDNA hubs in a JQ1-sensitive manner. Systematic silencing of ecDNA enhancers by CRISPR interference reveals intermolecular enhancer-gene activation among multiple oncogene loci that are amplified on distinct ecDNAs. Thus, protein-tethered ecDNA hubs enable intermolecular transcriptional regulation and may serve as units of oncogene function and cooperative evolution and as potential targets for cancer therapy.
    DOI:  https://doi.org/10.1038/s41586-021-04116-8
  24. Nat Commun. 2021 Nov 26. 12(1): 6778
    An atlas of protein turnover rates in mouse tissues.
    Zach Rolfs, Brian L Frey, Xudong Shi, Yoshitaka Kawai, Lloyd M Smith, Nathan V Welham.
      Protein turnover is critical to cellular physiology as well as to the growth and maintenance of tissues. The unique synthesis and degradation rates of each protein help to define tissue phenotype, and knowledge of tissue- and protein-specific half-lives is directly relevant to protein-related drug development as well as the administration of medical therapies. Using stable isotope labeling and mass spectrometry, we determine the in vivo turnover rates of thousands of proteins-including those of the extracellular matrix-in a set of biologically important mouse tissues. We additionally develop a data visualization platform, named ApplE Turnover, that enables facile searching for any protein of interest in a tissue of interest and then displays its half-life, confidence interval, and supporting measurements. This extensive dataset and the corresponding visualization software provide a reference to guide future studies of mammalian protein turnover in response to physiologic perturbation, disease, or therapeutic intervention.
    DOI:  https://doi.org/10.1038/s41467-021-26842-3
  25. J Clin Invest. 2021 Nov 23. pii: e149308. [Epub ahead of print]
    B-cell receptor isotypes differentially associate with cell signaling, kinetics, and outcome in chronic lymphocytic leukemia.
    Andrea N Mazzarello, Eva Gentner-Göbel, Marcus Dühren-von Minden, Tatyana N Tarasenko, Antonella Nicolò, Gerardo Ferrer, Stefano Vergani, Yun Liu, Davide Bagnara, Kanti R Rai, Jan A Burger, Peter J McGuire, Palash C Maity, Hassan Jumaa, Nicholas Chiorazzi.
      In chronic lymphocytic leukemia (CLL), the B-cell receptor (BCR) plays a critical role in disease development and progression as indicated by the therapeutic efficacy of drugs blocking BCR signaling. However, the mechanism(s) underlining BCR responsiveness are not completely defined. Selective engagement of membrane IgM or IgD on CLL cells, each co-expressed by > 90% of cases, leads to distinct signaling events. Since both IgM and IgD carry the same antigen-binding domains, the divergent actions of the receptors are attributed to differences in immunoglobulin (IG) structure or the outcome of signal transduction. We showed that IgM, not IgD, level and organization linked with CLL-cell birth rate and the type and consequences of BCR signaling in humans and mice. The latter IgM-driven effects were abrogated when BCR signaling was inhibited. Collectively, these studies demonstrated a critical, selective role for IgM in BCR signaling and B-cell fate decisions, possibly opening new avenues for CLL therapy.
    Keywords:  Immunoglobulins; Immunology; Leukemias; Oncology; Signal transduction
    DOI:  https://doi.org/10.1172/JCI149308
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