ATM Controls Genomic Stability in Pluripotent Stem Cells

Summary

Principal Investigator: JASON MARK BECKTA
Abstract: DESCRIPTION (provided by applicant): Pluripotent stem cells (PSCs) harbor exceptional potential for developing therapeutic advances for many diseases. However, when cultured in vitro, PSCs commonly acquire genetic abnormalities (e.g., aneuploidy). As these abnormalities can catalyze neoplastic progression, the genomic instability inherent to in vitro work has slowed efforts to bring PSCs from the bench to the bedside. Decatenation, the process of untangling chromosomes that become entwined during replication, contributes to the maintenance of genomic stability. Decatenation failure leads to a G2/M arrest via a distinct cell cycle checkpoint Activation of this checkpoint prevents aberrant daughter cell formation. It has been hypothesized that inefficient triggering of the decatenation checkpoint could be one cause of the observed genomic distortion. Recently, data has arisen demonstrating that the ataxia telangiectasia-mutated (ATM) kinase mediates this checkpoint, conflicting with older reports which posit that ATM has no part in executing cell cycle arrest in response to incomplete decatenation. ATM, a serine-threonine kinase, plays a major role in many intracellular signaling networks, such as the DNA damage response. Our lab has previously found that ATM, while present and active in PSCs, does not participate in canonical DNA damage repair pathways. Thus, we turned our attention to the decatenation checkpoint in order to elucidate ATM's role in PSCs. As we are in the process of developing induced pluripotent cells from normal and disease-specific fibroblasts, preliminary studies were conducted in human embryonic stem cells. Flow cytometry analysis showed that inhibition of decatenation activates ATM in PSCs and arrests them in G2. Inhibition of ATM using the highly-specific kinase inhibitor KU-60019 abrogates this arrest and allows PSCs to enter mitosis, resulting in bizarre chromosome morphology. Live cell imaging studies (accomplished preliminarily in non-transformed normal fibroblasts) revealed a prolonged metaphase-to-anaphase transition time after ATM inhibition. Combining inhibitors of decatenation and ATM resulted in failed karyokinesis and excessive anaphase bridge formation. After solidifying these findings, we will elucidate other proteins in the signaling cascade, specifically investigating if ATM interacts with Aurora-A and Plk1 while controlling this checkpoint. We will also determine if ATM inhibition for prolonged periods causes the development of aneuploidy using novel methods. In addition to its checkpoint role, there is evidence that ATM mediates apoptosis during certain stages of differentiation. As appropriate cell death is also important in preventing unwarranted growth, we aim to determine if ATM plays a role in apoptosis in PSCs. Western blotting and flow cytometry revealed that inhibition of ATM halts apoptosis at 3 and 6 hours, but increases it at 18 hours. Further investigation will clarify our preliminary studies. Studying specific protein interactions and mechanisms of genomic fidelity has led to the development of many chemotherapeutic agents and gives us insight into basic cellular operation. The goal of this proposal is to contribute to a greater understanding of processes that will impact future treatments and augment our understanding of cellular biochemistry.
Funding Period: 2012-08-25 - 2015-08-24
more information: NIH RePORT

Top Publications

  1. pmc Synthesis of water-soluble camptothecin-polyoxetane conjugates via click chemistry
    Olga Yu Zolotarskaya
    Department of Biomedical Engineering, Virginia Commonwealth University, Richmond, Virginia 23284, United States
    Mol Pharm 9:3403-8. 2012
  2. pmc ATM kinase inhibition preferentially sensitizes p53-mutant glioma to ionizing radiation
    Laura Biddlestone-Thorpe
    Department of Radiation Oncology, Massey Cancer Center, Virginia Commonwealth University, Richmond, Virginia 23112, USA
    Clin Cancer Res 19:3189-200. 2013
  3. pmc Revisiting p53 for cancer-specific chemo- and radiotherapy: ten years after
    Jason M Beckta
    Department of Radiation Oncology Virginia Commonwealth University Richmond, VA USA Department of Biochemistry and Molecular Biology Virginia Commonwealth University Richmond, VA USA
    Cell Cycle 13:710-3. 2014

Research Grants

  1. The Virtual Physiological Rat Project
    Daniel A Beard; Fiscal Year: 2013
  2. Center on the Microenvironment and Metastasis
    Michael L Shuler; Fiscal Year: 2013
  3. The MIT Center for Single-Cell Dynamics in Cancer (SCDC)
    Scott R Manalis; Fiscal Year: 2013
  4. MSKCC Center for Molecular Imaging in Cancer
    STEVEN MARK LARSON; Fiscal Year: 2013
  5. Structure and Function of DNA Repair Enzymes
    Susan S Wallace; Fiscal Year: 2013
  6. Center for Neuroplasticity at the University of Puerto Rico
    Steven N Treistman; Fiscal Year: 2013
  7. Cadiorenal and Metabolic Diseases Research Center
    John E Hall; Fiscal Year: 2013
  8. Signaling in Inflammation, Stress, and Tumorigenesis
    GEORGE ROBERT STARK; Fiscal Year: 2013
  9. Genomic stability of human pluripotent stem cells
    PAUL HUBERT LEROU; Fiscal Year: 2013
  10. Arterial Dysfunction: Basic and Clinical Mechanisms
    Thomas Michel; Fiscal Year: 2013

Detail Information

Publications3

  1. pmc Synthesis of water-soluble camptothecin-polyoxetane conjugates via click chemistry
    Olga Yu Zolotarskaya
    Department of Biomedical Engineering, Virginia Commonwealth University, Richmond, Virginia 23284, United States
    Mol Pharm 9:3403-8. 2012
    ..Altogether, we have synthesized CPT-polymer conjugates able to induce controlled toxicity to human cancer cells...
  2. pmc ATM kinase inhibition preferentially sensitizes p53-mutant glioma to ionizing radiation
    Laura Biddlestone-Thorpe
    Department of Radiation Oncology, Massey Cancer Center, Virginia Commonwealth University, Richmond, Virginia 23112, USA
    Clin Cancer Res 19:3189-200. 2013
    ..As a first step toward this goal, we recently showed that the novel ATM kinase inhibitor KU-60019 reduced migration, invasion, and growth, and potently radiosensitized human glioma cells in vitro...
  3. pmc Revisiting p53 for cancer-specific chemo- and radiotherapy: ten years after
    Jason M Beckta
    Department of Radiation Oncology Virginia Commonwealth University Richmond, VA USA Department of Biochemistry and Molecular Biology Virginia Commonwealth University Richmond, VA USA
    Cell Cycle 13:710-3. 2014
    ..This short review highlights several of these recent studies, discusses possible mechanism(s) for mp53-mediated "synthetic lethality," and the implications for cancer therapy. ..

Research Grants30

  1. The Virtual Physiological Rat Project
    Daniel A Beard; Fiscal Year: 2013
    ..This proposal targets the grand challenge of understanding complex multi-faceted disease phenotypes through experiments and simulations that capture the complex genotype-environment-phenotype relationship. ..
  2. Center on the Microenvironment and Metastasis
    Michael L Shuler; Fiscal Year: 2013
    ..By this approach the impact of this research should be felt far more widely than ordinary individual investigator projects. ..
  3. The MIT Center for Single-Cell Dynamics in Cancer (SCDC)
    Scott R Manalis; Fiscal Year: 2013
    ..These facilities and all reagents generated by the cores will be made available to other PS-OCs. ..
  4. MSKCC Center for Molecular Imaging in Cancer
    STEVEN MARK LARSON; Fiscal Year: 2013
    ..In summary, we have shaped ICMIC-3 to closely adhere to the goals of PAR 09-157. ..
  5. Structure and Function of DNA Repair Enzymes
    Susan S Wallace; Fiscal Year: 2013
    ..In addition to bioinformatics for all projects, Core A will also perform kinetics analysis for Projects 2-4. Core C will provide the administrative underpinnings for the project. ..
  6. Center for Neuroplasticity at the University of Puerto Rico
    Steven N Treistman; Fiscal Year: 2013
    ..This UPR COBRE Center should define pathways and benchmarks for basic and translational research across the UPR system for the next decades. ..
  7. Cadiorenal and Metabolic Diseases Research Center
    John E Hall; Fiscal Year: 2013
    ..abstract_text> ..
  8. Signaling in Inflammation, Stress, and Tumorigenesis
    GEORGE ROBERT STARK; Fiscal Year: 2013
    ..abstract_text> ..
  9. Genomic stability of human pluripotent stem cells
    PAUL HUBERT LEROU; Fiscal Year: 2013
    ..The goal of this research is to study the mechanisms of genomic stability maintenance in pluripotent cells in order to better understand the molecular basis of aneuploidy. ..
  10. Arterial Dysfunction: Basic and Clinical Mechanisms
    Thomas Michel; Fiscal Year: 2013
    ..Gladyshev. P. Libby directs the Redox Biomarkers Core;metabolic characterizations of mouse models studied in this Program will take place at the Yale Mouse Metabolic Phenotyping Center, led by G. Shulman. ..
  11. Spatial and Temporal Regulation of Angiogenesis
    HAROLD FISHER DVORAK; Fiscal Year: 2013
    ..abstract_text> ..
  12. Host-tumor cell interaction in myeloma: therapeutic applications
    Kenneth C Anderson; Fiscal Year: 2013
    ..Ultimately, our goal is to validate MM-host cell interactions as a target for novel therapeutics to improve patient outcome in MM. ..
  13. CSHL CANCER CENTER SUPPORT GRANT
    BRUCE W STILLMAN; Fiscal Year: 2013
    ....