bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2022–04–24
25 papers selected by
Ralitsa Radostinova Madsen, University College London
  1. The oncogenic PI3K-induced transcriptomic landscape reveals key functions in splicing and gene expression regulation.
  2. Assembly of nuclear dimers of PI3K regulatory subunits is regulated by the Cdc42-activated tyrosine kinase ACK.
  3. PI(4,5)P2 controls slit diaphragm formation and endocytosis in Drosophila nephrocytes.
  4. AKT mutant allele-specific activation dictates pharmacologic sensitivities.
  5. Signaling Pathways That Mediate Alveolar Macrophage Activation by Surfactant Protein A and IL-4.
  6. Inhibition of BTK and PI3Kδ impairs the development of Human JMML Stem and Progenitor cells.
  7. Computational study reveals substituted benzimidazole derivatives' binding selectivity to PI3Kδ and PI3Kγ.
  8. A Review on Cutaneous and Musculoskeletal Manifestations of CLOVES Syndrome.
  9. Genetic analysis of daf-18/PTEN missense mutants for starvation resistance and developmental regulation during Caenorhabditis elegans L1 arrest.
  10. Escargot controls somatic stem cell maintenance through the attenuation of the insulin receptor pathway in Drosophila.
  11. Distinct and additive effects of calorie restriction and rapamycin in aging skeletal muscle.
  12. mTORC1 Regulates High Levels of Protein Synthesis in Retinal Ganglion Cells of Adult Mice.
  13. Insulin action and resistance are dependent on a GSK3β-FBXW7-ERRα transcriptional axis.
  14. A preclinical platform for assessing antitumor effects and systemic toxicities of cancer drug targets.
  15. Late-onset Proteus syndrome with cerebriform connective tissue nevus and subsequent development of intraductal papilloma.
  16. Tetracycline response element driven Cre causes ectopic recombinase activity independent of transactivator element.
  17. Distinct resistance mechanisms arise to allosteric vs. ATP-competitive AKT inhibitors.
  18. Leukemia inhibitory factor drives glucose metabolic reprogramming to promote breast tumorigenesis.
  19. Retraction: Akt Regulates Drug-Induced Cell Death through Bcl-w Downregulation.
  20. The miR-26 family regulates early B cell development and transformation.
  21. Three live-imaging techniques for comprehensively understanding the initial trigger for insulin-responsive intracellular GLUT4 trafficking.
  22. Genetic analysis of DAF-18/PTEN missense mutants for the ability to maintain quiescence of the somatic gonad and germ line in C. elegans dauer larvae.
  23. Delivering precision oncology to patients with cancer.
  24. Krebs and an alternative TCA cycle!
  25. A loss-of-adhesion CRISPR-Cas9 screening platform to identify cell adhesion-regulatory proteins and signaling pathways.
  1. Cancer Res. 2022 Apr 20. pii: canres.CAN-22-0446-E.2022. [Epub ahead of print]
    The oncogenic PI3K-induced transcriptomic landscape reveals key functions in splicing and gene expression regulation.
    Erik Ladewig, Flavia Michelini, Komal Jhaveri, Pau Castel, Javier Carmona, Lauren Fairchild, Adler G Zuniga, Amaia Arruabarrena-Aristorena, Emiliano Cocco, Ryan Blawski, Srushti Kittane, Yuhan Zhang, Mirna Sallaku, Laura Baldino, Vasilis Hristidis, Sarat Chandarlapaty, Omar Abdel-Wahab, Christina Leslie, Maurizio Scaltriti, Eneda Toska.
      The PI3K pathway regulates proliferation, survival, and metabolism and is frequently activated across human cancers. A comprehensive elucidation of how this signaling pathway controls transcriptional and co-transcriptional processes could provide new insights into the key functions of PI3K signaling in cancer. Here, we undertook a transcriptomic approach to investigate genome-wide gene expression and transcription factor (TF) activity changes, as well as splicing and isoform usage dynamics, downstream of PI3K. These analyses uncovered widespread alternatively spliced (AS) isoforms linked to proliferation, metabolism, and splicing in PIK3CA mutant cells, which were reversed by inhibition of PI3Kα. Analysis of paired tumor biopsies from PIK3CA-mutated breast cancer patients undergoing treatment with PI3Kα inhibitors identified widespread splicing alterations that affect specific isoforms in common with the preclinical models, and these alterations, namely PTK2/FRNK and AFMID isoforms, were validated as functional drivers of cancer cell growth or migration. Mechanistically, isoform-specific splicing factors mediated PI3K-dependent RNA splicing. Treatment with splicing inhibitors rendered breast cancer cells more sensitive to the PI3Kα inhibitor alpelisib, resulting in greater growth inhibition than alpelisib alone. This study provides the first comprehensive analysis of widespread splicing alterations driven by oncogenic PI3K in breast cancer. The atlas of PI3K-mediated splicing programs establishes a key role for the PI3K pathway in regulating splicing, opening new avenues for exploiting PI3K signaling as a therapeutic vulnerability in breast cancer.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-22-0446
  2. J Biol Chem. 2022 Apr 13. pii: S0021-9258(22)00356-8. [Epub ahead of print] 101916
    Assembly of nuclear dimers of PI3K regulatory subunits is regulated by the Cdc42-activated tyrosine kinase ACK.
    Natasha S Clayton, Millie Fox, Jose J Vicenté-Garcia, Courtney M Schroeder, Trevor D Littlewood, Jonathon I Wilde, Kadalmani Krishnan, Murray J B Brown, Claire Crafter, Helen R Mott, Darerca Owen.
      Activated Cdc42-associated kinase (ACK) is an oncogenic non-receptor tyrosine kinase associated with poor prognosis in several human cancers. ACK promotes proliferation, in part by contributing to the activation of Akt, the major effector of class 1A phosphoinositide 3-kinases (PI3Ks), which transduce signals via membrane phosphoinositol lipids. We now show that ACK also interacts with other key components of class 1A PI3K signaling, the PI3K regulatory subunits. We demonstrate ACK binds to all five PI3K regulatory subunit isoforms and directly phosphorylates p85α, p85β, p50α, and p55α on Tyr607 (or analogous residues). We found that phosphorylation of p85β promotes cell proliferation in HEK293T cells. We demonstrate that ACK interacts with p85α exclusively in nuclear-enriched cell fractions, where p85α phosphorylated at Tyr607 (pTyr607) also resides, and identify an interaction between pTyr607 and the N-terminal SH2 domain that supports dimerization of the regulatory subunits. We infer from this that ACK targets p110-independent p85, and further postulate that these regulatory subunit dimers undertake novel nuclear functions underpinning ACK activity. We conclude that these dimers represent a previously undescribed mode of regulation for the class1A PI3K regulatory subunits and potentially reveal additional avenues for therapeutic intervention.
    Keywords:  Activated Cdc42 kinase; Cdc42; PI3Kinase; cancer; nuclear signalling; p110-independent p85; p85 dimers; protein degradation; protein phosphorylation; tyrosine kinase
    DOI:  https://doi.org/10.1016/j.jbc.2022.101916
  3. Cell Mol Life Sci. 2022 Apr 18. 79(5): 248
    PI(4,5)P2 controls slit diaphragm formation and endocytosis in Drosophila nephrocytes.
    Maximilian M Gass, Sarah Borkowsky, Marie-Luise Lotz, Rebecca Siwek, Rita Schröter, Pavel Nedvetsky, Stefan Luschnig, Astrid Rohlmann, Markus Missler, Michael P Krahn.
      Drosophila nephrocytes are an emerging model system for mammalian podocytes and proximal tubules as well as for the investigation of kidney diseases. Like podocytes, nephrocytes exhibit characteristics of epithelial cells, but the role of phospholipids in polarization of these cells is yet unclear. In epithelia, phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) and phosphatidylinositol(3,4,5)-trisphosphate (PI(3,4,5)P3) are asymmetrically distributed in the plasma membrane and determine apical-basal polarity. Here, we demonstrate that both phospholipids are present in the plasma membrane of nephrocytes, but only PI(4,5)P2 accumulates at slit diaphragms. Knockdown of Skittles, a phosphatidylinositol(4)phosphate 5-kinase, which produces PI(4,5)P2, abolished slit diaphragm formation and led to strongly reduced endocytosis. Notably, reduction in PI(3,4,5)P3 by overexpression of PTEN or expression of a dominant-negative phosphatidylinositol-3-kinase did not affect nephrocyte function, whereas enhanced formation of PI(3,4,5)P3 by constitutively active phosphatidylinositol-3-kinase resulted in strong slit diaphragm and endocytosis defects by ectopic activation of the Akt/mTOR pathway. Thus, PI(4,5)P2 but not PI(3,4,5)P3 is essential for slit diaphragm formation and nephrocyte function. However, PI(3,4,5)P3 has to be tightly controlled to ensure nephrocyte development.
    Keywords:  Nephrocyte; PI3-kinase; PTEN; Phosphoinositides; Phospholipids; Podocyte; Slit diaphragm
    DOI:  https://doi.org/10.1007/s00018-022-04273-7
  4. Nat Commun. 2022 Apr 19. 13(1): 2111
    AKT mutant allele-specific activation dictates pharmacologic sensitivities.
    Tripti Shrestha Bhattarai, Tambudzai Shamu, Alexander N Gorelick, Matthew T Chang, Debyani Chakravarty, Elena I Gavrila, Mark T A Donoghue, JianJong Gao, Swati Patel, Sizhi Paul Gao, Margaret H Reynolds, Sarah M Phillips, Tara Soumerai, Wassim Abida, David M Hyman, Alison M Schram, David B Solit, Lillian M Smyth, Barry S Taylor.
      AKT- a key molecular regulator of PI-3K signaling pathway, is somatically mutated in diverse solid cancer types, and aberrant AKT activation promotes altered cancer cell growth, survival, and metabolism1-8. The most common of AKT mutations (AKT1 E17K) sensitizes affected solid tumors to AKT inhibitor therapy7,8. However, the pathway dependence and inhibitor sensitivity of the long tail of potentially activating mutations in AKT is poorly understood, limiting our ability to act clinically in prospectively characterized cancer patients. Here we show, through population-scale driver mutation discovery combined with functional, biological, and therapeutic studies that some but not all missense mutations activate downstream AKT effector pathways in a growth factor-independent manner and sensitize tumor cells to diverse AKT inhibitors. A distinct class of small in-frame duplications paralogous across AKT isoforms induce structural changes different than those of activating missense mutations, leading to a greater degree of membrane affinity, AKT activation, and cell proliferation as well as pathway dependence and hyper-sensitivity to ATP-competitive, but not allosteric AKT inhibitors. Assessing these mutations clinically, we conducted a phase II clinical trial testing the AKT inhibitor capivasertib (AZD5363) in patients with solid tumors harboring AKT alterations (NCT03310541). Twelve patients were enrolled, out of which six harbored AKT1-3 non-E17K mutations. The median progression free survival (PFS) of capivasertib therapy was 84 days (95% CI 50-not reached) with an objective response rate of 25% (n = 3 of 12) and clinical benefit rate of 42% (n = 5 of 12). Collectively, our data indicate that the degree and mechanism of activation of oncogenic AKT mutants vary, thereby dictating allele-specific pharmacological sensitivities to AKT inhibition.
    DOI:  https://doi.org/10.1038/s41467-022-29638-1
  5. Front Immunol. 2022 ;13 860262
    Signaling Pathways That Mediate Alveolar Macrophage Activation by Surfactant Protein A and IL-4.
    Belén García-Fojeda, Carlos M Minutti, Carlos Montero-Fernández, Cordula Stamme, Cristina Casals.
      Activation of tissue repair program in macrophages requires the integration of IL-4/IL-13 cytokines and tissue-specific signals. In the lung, surfactant protein A (SP-A) is a tissue factor that amplifies IL-4Rα-dependent alternative activation and proliferation of alveolar macrophages (AMs) through the myosin18A receptor. However, the mechanism by which SP-A and IL-4 synergistically increase activation and proliferation of AMs is unknown. Here we show that SP-A amplifies IL-4-mediated phosphorylation of STAT6 and Akt by binding to myosin18A. Blocking PI3K activity or the myosin18A receptor abrogates SP-A´s amplifying effects on IL-4 signaling. SP-A alone activates Akt, mTORC1, and PKCζ and inactivates GSK3α/β by phosphorylation, but it cannot activate arginase-1 activity or AM proliferation on its own. The combined effects of IL-4 and SP-A on the mTORC1 and GSK3 branches of PI3K-Akt signaling contribute to increased AM proliferation and alternative activation, as revealed by pharmacological inhibition of Akt (inhibitor VIII) and mTORC1 (rapamycin and torin). On the other hand, the IL-4+SP-A-driven PKCζ signaling axis appears to intersect PI3K activation with STAT6 phosphorylation to achieve more efficient alternative activation of AMs. Consistent with IL-4+SP-A-driven activation of mTORC1 and mTORC2, both agonists synergistically increased mitochondrial respiration and glycolysis in AMs, which are necessary for production of energy and metabolic intermediates for proliferation and alternative activation. We conclude that SP-A signaling in AMs activates PI3K-dependent branched pathways that amplify IL-4 actions on cell proliferation and the acquisition of AM effector functions.
    Keywords:  IL-4; PKCζ; Pi3k-akt; mTORC1; macrophage alternative activation; metabolism; proliferation; surfactant protein A
    DOI:  https://doi.org/10.3389/fimmu.2022.860262
  6. Mol Ther. 2022 Apr 19. pii: S1525-0016(22)00238-6. [Epub ahead of print]
    Inhibition of BTK and PI3Kδ impairs the development of Human JMML Stem and Progenitor cells.
    Baskar Ramdas, Lisa Deng Yuen, Lakshmi Reddy Palam, Roshini Patel, Santhosh Kumar Pasupuleti, Victoria Jideonwo, Ji Zhang, Callista Maguire, Eric Wong, Rahul Kanumuri, Chujing Zhang, George Sandusky, Rebecca J Chan, Chi Zhang, Elliot Stieglitz, Laura Haneline, Reuben Kapur.
      JMML is an aggressive Myeloproliferative neoplasia (MPN) that lacks effective targeted chemotherapies. Clinically, JMML manifests as monocytic leukocytosis, splenomegaly with consequential thrombocytopenia. Most commonly, patients have gain-of-function (GOF) oncogenic mutations in PTPN11 (SHP2), leading to Erk and Akt hyperactivation. Mechanism(s) involved in co-regulation of Erk and Akt in the context of GOF SHP2 are poorly understood. Here, we show that Bruton's tyrosine kinase (BTK) is hyperphosphorylated in GOF Shp2 bearing cells and utilizes B cell adaptor for PI3K (BCAP) to cooperate with p110δ, the catalytic subunit of PI3K. Dual inhibition of BTK and p110δ reduces the activation of both Erk and Akt. In vivo, individual targeting of BTK or p110δ in a mouse model of human JMML equally reduces monocytosis and splenomegaly, however, the combined treatment results in a more robust inhibition and uniquely rescues anemia and thrombocytopenia. RNAseq analysis of drug treated mice showed a profound reduction in the expression of genes associated with leukemic cell migration and inflammation, leading to correction in the infiltration of leukemic cells in the lung, liver and spleen. Remarkably, in a patient derived xenograft model of JMML, leukemia initiating stem and progenitor cells were potently inhibited in response to the dual drug treatment.
    DOI:  https://doi.org/10.1016/j.ymthe.2022.04.009
  7. J Mol Model. 2022 Apr 19. 28(5): 123
    Computational study reveals substituted benzimidazole derivatives' binding selectivity to PI3Kδ and PI3Kγ.
    Na-Na Zhang, Xue Bai, Shan-Shan Zhao, Xue-Mei Zheng, Lei Tang, Sheng-Gang Yang, Ji-Quan Zhang.
      Phosphatidylinositol 3-kinase (PI3K) is a key regulatory kinase in the PI3K/AKT/mTOR signaling pathway, which is involved in the regulation of cell proliferation, differentiation, apoptosis, and angiogenesis. Class IA PI3K isoforms γ and δ share a highly homologous ATP binding site and are distinguished by only a few residues around the binding site. Subtype-selective inhibitors have been proven to have great advantages in tumor treatment. Preliminary studies have obtained PI3K inhibitors bearing a benzimidazole structural motif with a certain selectivity for PI3Kδ and PI3Kγ subtypes. On this basis, we investigated the selective inhibitory mechanism of PI3Kδ and PI3Kγ using four developed inhibitors via molecular docking, molecular dynamics, binding free energy calculations, and residue energy decomposition. This study could provide references for the further development of PI3K-isoform-selective inhibitors.
    Keywords:  Isoform selective inhibitors; MMGBSA; Molecular docking; Molecular dynamics; Phosphatidylinositol 3-kinase
    DOI:  https://doi.org/10.1007/s00894-022-05096-w
  8. Clin Cosmet Investig Dermatol. 2022 ;15 621-630
    A Review on Cutaneous and Musculoskeletal Manifestations of CLOVES Syndrome.
    Emel Öztürk Durmaz, Deniz Demircioğlu, Pınar Yalınay Dikmen, Yasemin Alanay, Ahmet Alanay, Cüyan Demirkesen, Fatma Tokat, Ercan Karaarslan.
      CLOVES syndrome is a novel sporadic mosaic segmental overgrowth syndrome, currently categorized under the canopy of PROS (PIK3CA-related overgrowth spectrum) disorders. All PROS disorders harbor heterozygous postzygotic activating somatic mutations involving the PIK3CA gene. As an upstream regulator of the PI3K/AKT/mTOR signal transduction pathway, activating mutations of PIK3CA gene commence in uncontrolled growth of cutaneous, vascular (capillaries, veins, and lymphatics), adipose, neural, and musculoskeletal tissues. The excessive growth is segmental, patchy, asymmetric, and confined to body parts affected by the mutation. The term 'CLOVES' is an acronym denoting congenital lipomatous overgrowth, vascular malformations, epidermal nevi and spinal (scoliosis) and/ or skeletal anomalies. The syndrome is characterized by an admixture of overgrown tissues, derived mainly from mesoderm and neuroectoderm. Among PROS disorders, CLOVES syndrome represents the extreme end of the spectrum with massive affection of almost the entire body. The syndrome might judiciously be treated with medications hampering with the PI3K/AKT/mTOR signal transduction pathway. This article aims at reviewing the cutaneous and musculoskeletal manifestations of CLOVES syndrome, as the paradigm for PROS disorders. CLOVES syndrome and other PROS disorders are still misdiagnosed, underdiagnosed, underreported, and undertreated by the dermatology community.
    Keywords:  CLOVES syndrome; PIK3CA-related overgrowth spectrum; cutaneous manifestations; epidermal nevi; lipomas; lymphangiomas; port wine stains
    DOI:  https://doi.org/10.2147/CCID.S351637
  9. G3 (Bethesda). 2022 Apr 22. pii: jkac092. [Epub ahead of print]
    Genetic analysis of daf-18/PTEN missense mutants for starvation resistance and developmental regulation during Caenorhabditis elegans L1 arrest.
    Jingxian Chen, Linda Y Tang, Maya E Powell, James M Jordan, L Ryan Baugh.
      Mutations in the well-known tumor suppressor PTEN are observed in many cancers. PTEN is a dual-specificity phosphatase that harbors lipid and protein-phosphatase activities. The Caenorhabditis elegans PTEN ortholog is daf-18, which has pleiotropic effects on dauer formation, aging, starvation resistance, and development. Function of three daf-18 point-mutants, G174E, D137A and C169S, had previously been investigated using high-copy transgenes in a daf-18 null background. These alleles were generated based on their mammalian counterparts and were treated as though they specifically disrupt lipid or protein-phosphatase activity, or both, respectively. Here, we investigated these alleles using genome editing of endogenous daf-18. We assayed three traits relevant to L1 starvation resistance, and we show that each point mutant is essentially as starvation-sensitive as a daf-18 null mutant. Furthermore, we show that G174E and D137A do not complement each other, suggesting overlapping effects on lipid and protein-phosphatase activity. We also show that each allele has strong effects on nucleocytoplasmic localization of DAF-16/FoxO and dauer formation, both of which are regulated by PI3K signaling, similar to a daf-18 null allele. In addition, each allele also disrupts M-cell quiescence during L1 starvation, though D137A has a weaker effect than the other alleles, including the null. Our results confirm that daf-18/PTEN is important for promoting starvation resistance and developmental arrest and that it is a potent regulator of PI3K signaling, and they highlight challenges of using genetic analysis to link specific DAF-18/PTEN enzymatic activities to particular phenotypes.
    Keywords:  DAF-18; L1 arrest; L1 diapause; PTEN; quiescence; starvation
    DOI:  https://doi.org/10.1093/g3journal/jkac092
  10. Cell Rep. 2022 Apr 19. pii: S2211-1247(22)00431-4. [Epub ahead of print]39(3): 110679
    Escargot controls somatic stem cell maintenance through the attenuation of the insulin receptor pathway in Drosophila.
    Rafael Sênos Demarco, Brian J Stack, Alexander M Tang, Justin Voog, Sharsti L Sandall, Tony D Southall, Andrea H Brand, D Leanne Jones.
      Adult stem cells coordinate intrinsic and extrinsic, local and systemic, cues to maintain the proper balance between self-renewal and differentiation. However, the precise mechanisms stem cells use to integrate these signals remain elusive. Here, we show that Escargot (Esg), a member of the Snail family of transcription factors, regulates the maintenance of somatic cyst stem cells (CySCs) in the Drosophila testis by attenuating the activity of the pro-differentiation insulin receptor (InR) pathway. Esg positively regulates the expression of an antagonist of insulin signaling, ImpL2, while also attenuating the expression of InR. Furthermore, Esg-mediated repression of the InR pathway is required to suppress CySC loss in response to starvation. Given the conservation of Snail-family transcription factors, characterizing the mechanisms by which Esg regulates cell-fate decisions during homeostasis and a decline in nutrient availability is likely to provide insight into the metabolic regulation of stem cell behavior in other tissues and organisms.
    Keywords:  CP: Developmental biology; CP: Stem cell research; Drosophila; ImpL2; InR; cyst stem cells; escargot; insulin; metabolism; testis
    DOI:  https://doi.org/10.1016/j.celrep.2022.110679
  11. Nat Commun. 2022 Apr 19. 13(1): 2025
    Distinct and additive effects of calorie restriction and rapamycin in aging skeletal muscle.
    Daniel J Ham, Anastasiya Börsch, Kathrin Chojnowska, Shuo Lin, Aurel B Leuchtman, Alexander S Ham, Marco Thürkauf, Julien Delezie, Regula Furrer, Dominik Burri, Michael Sinnreich, Christoph Handschin, Lionel A Tintignac, Mihaela Zavolan, Nitish Mittal, Markus A Rüegg.
      Preserving skeletal muscle function is essential to maintain life quality at high age. Calorie restriction (CR) potently extends health and lifespan, but is largely unachievable in humans, making "CR mimetics" of great interest. CR targets nutrient-sensing pathways centering on mTORC1. The mTORC1 inhibitor, rapamycin, is considered a potential CR mimetic and is proven to counteract age-related muscle loss. Therefore, we tested whether rapamycin acts via similar mechanisms as CR to slow muscle aging. Here we show that long-term CR and rapamycin unexpectedly display distinct gene expression profiles in geriatric mouse skeletal muscle, despite both benefiting aging muscles. Furthermore, CR improves muscle integrity in mice with nutrient-insensitive, sustained muscle mTORC1 activity and rapamycin provides additive benefits to CR in naturally aging mouse muscles. We conclude that rapamycin and CR exert distinct, compounding effects in aging skeletal muscle, thus opening the possibility of parallel interventions to counteract muscle aging.
    DOI:  https://doi.org/10.1038/s41467-022-29714-6
  12. J Biol Chem. 2022 Apr 18. pii: S0021-9258(22)00384-2. [Epub ahead of print] 101944
    mTORC1 Regulates High Levels of Protein Synthesis in Retinal Ganglion Cells of Adult Mice.
    Patrice E Fort, Mandy K Losiewicz, Lynda Elghazi, Dejuan Kong, Corentin Cras-Méneur, Diane C Fingar, Scot R Kimball, Raju V S Rajala, Alexander J Smith, Robin R Ali, Steven F Abcouwer, Thomas W Gardner.
      Mechanistic target of rapamycin (mTOR) and members of mTOR complex 1 (mTORC1), a linchpin of the nutrient sensing and protein synthesis pathways, are present at relatively high levels in the ganglion cell layer (GCL) and retinal ganglion cells (RGC) of rodent and human retinas. However, the role of mTOR complexes in the control of protein synthesis in RGC is unknown. Here we applied the SUnSET method of nascent protein labeling to localize and quantify protein synthesis in the retinas of adult mice. We also used intravitreal injection of an AAV2 vector encoding Cre recombinase in the eyes of mtor- or rptor-floxed mice to conditionally knockout either both mTOR complexes or only mTORC1, respectively, in cells within the GCL. A novel vector encoding an inactive Cre mutant (CreΔC) served as control. We found that retinal protein synthesis was highest in the GCL, particularly in RGC. Negation of both complexes or only mTORC1 significantly reduced protein synthesis in RGC. In addition, loss of mTORC1 function caused a significant reduction in the pan-RGC marker, RNA binding protein with multiple splicing (RBPMS), with little decrease of the total number of cells in the RGC layer, even at 25 weeks after AAV-Cre injection. These findings reveal that mTORC1 signaling is necessary for maintaining the high rate of protein synthesis in RGCs of adult rodents, but it may not be essential to maintain RGC viability. These findings may also be relevant to understanding the pathophysiology of RGC disorders, including glaucoma, diabetic retinopathy, and optic neuropathies.
    Keywords:  eye; gene knockout; mRNA translation; mTOR complex (mTORC); mTORC1; mechanistic target of rapamycin (mTOR); protein synthesis; puromycin; retina; retinal ganglion cell
    DOI:  https://doi.org/10.1016/j.jbc.2022.101944
  13. Nat Commun. 2022 Apr 19. 13(1): 2105
    Insulin action and resistance are dependent on a GSK3β-FBXW7-ERRα transcriptional axis.
    Hui Xia, Charlotte Scholtes, Catherine R Dufour, Carlo Ouellet, Majid Ghahremani, Vincent Giguère.
      Insulin resistance, a harbinger of the metabolic syndrome, is a state of compromised hormonal response resulting from the dysregulation of a wide range of insulin-controlled cellular processes. However, how insulin affects cellular energy metabolism via long-term transcriptional regulation and whether boosting mitochondrial function alleviates insulin resistance remains to be elucidated. Herein we reveal that insulin directly enhances the activity of the nuclear receptor ERRα via a GSK3β/FBXW7 signaling axis. Liver-specific deletion of GSK3β or FBXW7 and mice harboring mutations of ERRα phosphosites (ERRα3SA) co-targeted by GSK3β/FBXW7 result in accumulated ERRα proteins that no longer respond to fluctuating insulin levels. ERRα3SA mice display reprogrammed liver and muscle transcriptomes, resulting in compromised energy homeostasis and reduced insulin sensitivity despite improved mitochondrial function. This crossroad of insulin signaling and transcriptional control by a nuclear receptor offers a framework to better understand the complex cellular processes contributing to the development of insulin resistance.
    DOI:  https://doi.org/10.1038/s41467-022-29722-6
  14. Proc Natl Acad Sci U S A. 2022 Apr 26. 119(17): e2110557119
    A preclinical platform for assessing antitumor effects and systemic toxicities of cancer drug targets.
    Xiang Li, Chun-Hao Huang, Francisco J Sánchez-Rivera, Margaret C Kennedy, Darjus F Tschaharganeh, John P Morris, Antonella Montinaro, Kevin P O'Rourke, Ana Banito, John E Wilkinson, Chi-Chao Chen, Yu-Jui Ho, Lukas E Dow, Sha Tian, Wei Luan, Elisa de Stanchina, Tinghu Zhang, Nathanael S Gray, Henning Walczak, Scott W Lowe.
      SignificanceMany new cancer drugs fail at the clinical stage owing to poor efficacy and/or excessive toxicity, though whether this reflects shortcomings of the target or the drug is often unclear. To gain earlier insights into factors that can influence the therapeutic index of target inhibition in vivo, we combine inducible RNA interference and somatic engineering technologies to produce a cost-effective platform that enables systemic and inducible suppression of candidate target in normal tissues and tumor cells in the same mouse. By comparing the consequences of genetic and pharmacological CDK9 inhibition, we establish the utility of this platform to predict factors influencing the therapeutic index. Additionally, our studies provide support, and some cautionary notes, for the clinical development of CDK9 inhibitors.
    Keywords:  CDK9; hepatocellular carcinoma; mouse model; preclinical platform
    DOI:  https://doi.org/10.1073/pnas.2110557119
  15. Am J Med Genet A. 2022 Apr 20.
    Late-onset Proteus syndrome with cerebriform connective tissue nevus and subsequent development of intraductal papilloma.
    Emily W Modlin, Anne M Slavotinek, Thomas N Darling, Stanley Lipkowitz, Frederic G Barr, Pamela N Munster, Leslie G Biesecker, Christopher A Ours.
      Proteus syndrome (PS) is a rare segmental overgrowth disorder caused by a mosaic activating variant in AKT1. The features of PS are often not present at birth but develop during the first few years of life. We describe a 55-year-old female, whose first symptom of overgrowth, a cerebriform connective tissue nevus, occurred at 19 years of age. We report the identification of the AKT1 c.49G > A p.(Glu17Lys) variant in this progressive lesion, the bony overgrowth, and recurrence after surgical intervention. In the sixth decade of life, this individual developed intraductal papillomas within her right breast which were confirmed to contain the same activating AKT1 variant as the connective tissue nevus. While similar neoplasms have been described in an individual with Proteus syndrome, none has been evaluated for the presence of the AKT1 variant. The tumor also contained two likely pathogenic variants in PIK3R1, c.1392_1403dupTAGATTATATGA p.(Asp464_Tyr467dup) and c.1728_1730delGAG p.(Arg577del). The finding of additional genetic variation putatively affecting the PI3K/AKT pathway in the neoplastic tissue may provide preliminary evidence of a molecular mechanism for tumorigenesis in PS. The late onset of symptoms and molecular characterization of the breast tumor expand the clinical spectrum of this rare disorder.
    Keywords:  Proteus syndrome; connective tissue nevus; intraductal papilloma; overgrowth
    DOI:  https://doi.org/10.1002/ajmg.a.62761
  16. Mol Metab. 2022 Apr 19. pii: S2212-8778(22)00070-9. [Epub ahead of print] 101501
    Tetracycline response element driven Cre causes ectopic recombinase activity independent of transactivator element.
    Kenneth T Lewis, Lily R Oles, Ormond A MacDougald.
       OBJECTIVE: Tamoxifen is widely used for inducible Cre-LoxP systems but has several undesirable side effects for researchers investigating metabolism or energy balance, including weight loss, lipoatrophy, and drug incorporation into lipid stores. For this reason, we sought to determine whether a doxycycline-inducible system would be more advantageous for adipocyte-specific Cre mouse models, but serendipitously discovered widespread ectopic tetracycline response element Cre (TRE-Cre) recombinase activity.
    METHODS: Adipocyte-specific tamoxifen- and doxycycline-inducible Cre mice were crossed to fluorescent Cre reporter mice and visualized by confocal microscopy to assess efficiency and background activity. TRE-Cre mice were crossed to stop-floxed diphtheria toxin mice to selectively ablate cells with background Cre activity.
    RESULTS: Tamoxifen- and doxycycline-inducible systems performed similarly in adipose tissues, but ectopic Cre recombination was evident in numerous other cell types of the latter, most notably neurons. The source of ectopic Cre activity was isolated to the TRE-Cre transgene, driven by the pTet (tetO7) tetracycline-inducible promoter. Ablation of cells with ectopic recombination in mice led to stunted growth, diminished survival, and reduced brain mass.
    CONCLUSIONS: These results indicate that tamoxifen- and doxycycline-inducible adipocyte-specific Cre mouse models are similarly efficient, but the TRE-Cre component of the latter is inherently leaky. TRE-Cre background activity is especially pronounced in the brain and peripheral nerve fibers, and selective ablation of these cells impairs mouse development and survival. Caution should be taken when pairing TRE-Cre with floxed alleles that have defined roles in neural function, and additional controls should be included when using this model system.
    Keywords:  AdipoChaser; Ectopic Cre activity; TRE-Cre; pTet; tetO7-Cre; tetracycline response element
    DOI:  https://doi.org/10.1016/j.molmet.2022.101501
  17. Nat Commun. 2022 Apr 19. 13(1): 2057
    Distinct resistance mechanisms arise to allosteric vs. ATP-competitive AKT inhibitors.
    Kristin M Zimmerman Savill, Brian B Lee, Jason Oeh, Jie Lin, Eva Lin, Wei-Jen Chung, Amy Young, Wennie Chen, Monika Miś, Kathryn Mesh, Jeffrey Eastham, Florian Gnad, Zhaoshi Jiang, Eric W Stawiski, Benjamin Haley, Anneleen Daemen, Xiaojing Wang, Hartmut Koeppen, Zora Modrusan, Scott E Martin, Deepak Sampath, Kui Lin.
      The AKT kinases have emerged as promising therapeutic targets in oncology and both allosteric and ATP-competitive AKT inhibitors have entered clinical investigation. However, long-term efficacy of such inhibitors will likely be challenged by the development of resistance. We have established prostate cancer models of acquired resistance to the allosteric inhibitor MK-2206 or the ATP-competitive inhibitor ipatasertib following prolonged exposure. While alterations in AKT are associated with acquired resistance to MK-2206, ipatasertib resistance is driven by rewired compensatory activity of parallel signaling pathways. Importantly, MK-2206 resistance can be overcome by treatment with ipatasertib, while ipatasertib resistance can be reversed by co-treatment with inhibitors of pathways including PIM signaling. These findings demonstrate that distinct resistance mechanisms arise to the two classes of AKT inhibitors and that combination approaches may reverse resistance to ATP-competitive inhibition.
    DOI:  https://doi.org/10.1038/s41467-022-29655-0
  18. Cell Death Dis. 2022 Apr 19. 13(4): 370
    Leukemia inhibitory factor drives glucose metabolic reprogramming to promote breast tumorigenesis.
    Xuetian Yue, Jianming Wang, Chun-Yuan Chang, Juan Liu, Xue Yang, Fan Zhou, Xia Qiu, Vrushank Bhatt, Jessie Yanxiang Guo, Xiaoyang Su, Lanjing Zhang, Zhaohui Feng, Wenwei Hu.
      LIF, a multifunctional cytokine, is frequently overexpressed in many types of solid tumors, including breast cancer, and plays an important role in promoting tumorigenesis. Currently, how LIF promotes tumorigenesis is not well-understood. Metabolic reprogramming is a hallmark of cancer cells and a key contributor to cancer progression. However, the role of LIF in cancer metabolic reprogramming is unclear. In this study, we found that LIF increases glucose uptake and drives glycolysis, contributing to breast tumorigenesis. Blocking glucose uptake largely abolishes the promoting effect of LIF on breast tumorigenesis. Mechanistically, LIF overexpression enhances glucose uptake via activating the AKT/GLUT1 axis to promote glycolysis. Blocking the AKT signaling by shRNA or its inhibitors greatly inhibits glycolysis driven by LIF and largely abolishes the promoting effect of LIF on breast tumorigenesis. These results demonstrate an important role of LIF overexpression in glucose metabolism reprogramming in breast cancers, which contributes to breast tumorigenesis. This study also reveals an important mechanism underlying metabolic reprogramming of breast cancers, and identifies LIF and its downstream signaling as potential therapeutic targets for breast cancers, especially those with LIF overexpression.
    DOI:  https://doi.org/10.1038/s41419-022-04820-x
  19. PLoS One. 2022 ;17(4): e0267621
    Retraction: Akt Regulates Drug-Induced Cell Death through Bcl-w Downregulation.
    PLOS ONE Editors
      
    DOI:  https://doi.org/10.1371/journal.pone.0267621
  20. Life Sci Alliance. 2022 Aug;pii: e202101303. [Epub ahead of print]5(8):
    The miR-26 family regulates early B cell development and transformation.
    Katharina Hutter, Silke E Lindner, Constanze Kurschat, Thomas Rülicke, Andreas Villunger, Sebastian Herzog.
      MiRNAs are small noncoding RNAs that promote the sequence-specific repression of their respective target genes, thereby regulating diverse physiological as well as pathological processes. Here, we identify a novel role of the miR-26 family in early B cell development. We show that enhanced expression of miR-26 family members potently blocks the pre-B to immature B cell transition, promotes pre-B cell expansion and eventually enables growth factor independency. Mechanistically, this is at least partially mediated by direct repression of the tumor-suppressor Pten, which consequently enhances PI3K-AKT signaling. Conversely, limiting miR-26 activity in a more physiological loss-of-function approach counteracts proliferation and enhances pre-B cell differentiation in vitro as well as in vivo. We therefore postulate a rheostat-like role for the miR-26 family in progenitor B cells, with an increase in mature miR-26 levels signaling cell expansion, and facilitating pre-B to the immature B cell progression when reduced.
    DOI:  https://doi.org/10.26508/lsa.202101303
  21. iScience. 2022 Apr 15. 25(4): 104164
    Three live-imaging techniques for comprehensively understanding the initial trigger for insulin-responsive intracellular GLUT4 trafficking.
    Hiroyasu Hatakeyama, Ko Kobayashi, Makoto Kanzaki.
      Quantitative features of GLUT4 glucose transporter's behavior deep inside cells remain largely unknown. Our previous analyses with live-cell imaging of intracellular GLUT4 trafficking demonstrated two crucial early events responsible for triggering insulin-responsive translocation processes, namely, heterotypic fusion and liberation. To quantify the regulation, interrelationships, and dynamics of the initial events more accurately and comprehensively, we herein applied three analyses, each based on our distinct dual-color live-cell imaging approaches. With these approaches, heterotypic fusion was found to be the first trigger for insulin-responsive GLUT4 redistributions, preceding liberation, and to be critically regulated by Akt substrate of 160 kDa (AS160) and actin dynamics. In addition, demonstrating the subcellular regional dependence of GLUT4 dynamics revealed that liberated GLUT4 molecules are promptly incorporated into the trafficking itinerary of transferrin receptors. Our approaches highlight the physiological significance of endosomal "GLUT4 molecule trafficking" rather than "GLUT4 vesicle delivery" to the plasma membrane in response to insulin.
    Keywords:  Biological sciences; Biological sciences research methodologies; Biology experimental methods; Cell biology; Optical imaging
    DOI:  https://doi.org/10.1016/j.isci.2022.104164
  22. G3 (Bethesda). 2022 Apr 22. pii: jkac093. [Epub ahead of print]
    Genetic analysis of DAF-18/PTEN missense mutants for the ability to maintain quiescence of the somatic gonad and germ line in C. elegans dauer larvae.
    Julia Wittes, Iva Greenwald.
      The mammalian tumor suppressor PTEN has well-established lipid phosphatase and protein phosphatase activities. DAF-18, the C. elegans ortholog of PTEN, has a high degree of conservation in the catalytic domain, and human PTEN complements a null allele of daf-18, suggesting conserved protein function. Insights gleaned from studies of mammalian PTEN have been applied to studies of DAF-18 in C. elegans, including predicted enzymatic properties of mutants. Here, we characterize DAF-18 missense mutants previously treated as selectively disrupting either protein or lipid phosphatase activity in genetic assays in order to connect distinct phenotypes to specific enzymatic activities of DAF-18/PTEN. We analyze the ability of these mutants to maintain quiescence of the somatic gonad and germ line in dauer larvae, a state of diapause during which development is suspended. We show that transgenes expressing either the putative lipid phosphatase-deficient or putative protein phosphatase-deficient form fail to complement a daf-18 null allele, and that the corresponding homozygous endogenous missense mutant alleles fail to maintain developmental quiescence. We also show that the endogenous daf-18 missense alleles fail to complement each other, suggesting that one or both of the missense forms are not activity-selective. Furthermore, homozygous daf-18 missense mutants cause a more severe phenotype than a daf-18 null mutant, suggesting the presence of functionally compromised mutant DAF-18 is more deleterious than the absence of DAF-18. We discuss how these genetic properties complicate the interpretation of genetic assays to associate specific enzymatic activities with specific phenotypes.
    Keywords:   C. elegans ; DAF-18; PTEN; dauer; gonad; quiescence
    DOI:  https://doi.org/10.1093/g3journal/jkac093
  23. Nat Med. 2022 Apr;28(4): 658-665
    Delivering precision oncology to patients with cancer.
    Joaquin Mateo, Lotte Steuten, Philippe Aftimos, Fabrice André, Mark Davies, Elena Garralda, Jan Geissler, Don Husereau, Iciar Martinez-Lopez, Nicola Normanno, Jorge S Reis-Filho, Stephen Stefani, David M Thomas, C Benedikt Westphalen, Emile Voest.
      With the increasing use of genomic profiling for diagnosis and therapy guidance in many tumor types, precision oncology is rapidly reshaping cancer care. However, the current trajectory of drug development in oncology results in a paradox: if patients cannot access advanced diagnostics, we may be developing drugs that will reach few patients. In this Perspective, we outline the major challenges to the implementation of precision oncology and discuss critical steps toward resolving these, including facilitation of equal access to genomics tests, ensuring that clinical studies provide robust evidence for new drugs and technologies, enabling physicians to interpret genomics data, and empowering patients toward shared decision-making. A multi-stakeholder approach to evidence generation, value assessment, and healthcare delivery is necessary to translate advances in precision oncology into benefits for patients with cancer globally.
    DOI:  https://doi.org/10.1038/s41591-022-01717-2
  24. Cell Res. 2022 Apr 22.
    Krebs and an alternative TCA cycle!
    Mary T Doan, Michael A Teitell.
      
    DOI:  https://doi.org/10.1038/s41422-022-00664-4
  25. Nat Commun. 2022 Apr 19. 13(1): 2136
    A loss-of-adhesion CRISPR-Cas9 screening platform to identify cell adhesion-regulatory proteins and signaling pathways.
    Martin F M de Rooij, Yvonne J Thus, Nathalie Swier, Roderick L Beijersbergen, Steven T Pals, Marcel Spaargaren.
      The clinical introduction of the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, which targets B-cell antigen-receptor (BCR)-controlled integrin-mediated retention of malignant B cells in their growth-supportive lymphoid organ microenvironment, provided a major breakthrough in lymphoma and leukemia treatment. Unfortunately, a significant subset of patients is intrinsically resistant or acquires resistance against ibrutinib. Here, to discover novel therapeutic targets, we present an unbiased loss-of-adhesion CRISPR-Cas9 knockout screening method to identify proteins involved in BCR-controlled integrin-mediated adhesion. Illustrating the validity of our approach, several kinases with an established role in BCR-controlled adhesion, including BTK and PI3K, both targets for clinically applied inhibitors, are among the top hits of our screen. We anticipate that pharmacological inhibitors of the identified targets, e.g. PAK2 and PTK2B/PYK2, may have great clinical potential as therapy for lymphoma and leukemia patients. Furthermore, this screening platform is highly flexible and can be easily adapted to identify cell adhesion-regulatory proteins and signaling pathways for other stimuli, adhesion molecules, and cell types.
    DOI:  https://doi.org/10.1038/s41467-022-29835-y
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