title: MERTK Inhibition Induces Polyploidy and Promotes Cell Death and Cellular Senescence in Glioblastoma Multiforme.
abstract: BACKGROUND
  MER receptor tyrosine kinase (MERTK) is expressed in a variety of malignancies, including glioblastoma multiforme (GBM). Our previous work demonstrated that inhibition of MERTK using RNA interference induced cell death and chemosensitivity in GBM cells, implicating MERTK as a potential therapeutic target. Here we investigate whether a novel MERTK-selective small molecule tyrosine kinase inhibitor, UNC2025, has similar anti-tumor effects in GBM cell lines.
  
  
  METHODS
  Correlations between expression of GAS6, a MERTK ligand, and prognosis were determined using data from the TCGA database. GBM cell lines (A172, SF188, U251) were treated in vitro with increasing doses of UNC2025 (50-400nM). Cell count and viability were determined by trypan blue exclusion. Cell cycle profiles and induction of apoptosis were assessed by flow cytometric analysis after BrdU or Po-Pro-1/propidium iodide staining, respectively. Polyploidy was detected by propidium iodide staining and metaphase spread. Cellular senescence was determined by β-galactosidase staining and senescence-associated secretory cytokine analysis.
  
  
  RESULTS
  Decreased overall survival significantly correlated with high levels of GAS6 expression in GBM, highlighting the importance of TAM kinase signaling in GBM tumorigenesis and/or therapy resistance and providing strong rationale for targeting these pathways in the clinic. All three GBM cell lines exhibited dose dependent reductions in cell number and colony formation (>90% at 200nM) after treatment with UNC2025. Cell cycle analysis demonstrated accumulation of cells in the G2/M phase and development of polyploidy. After extended exposure, 60-80% of cells underwent apoptosis. The majority of surviving cells (65-95%) were senescent and did not recover after drug removal. Thus, UNC2025 mediates anti-tumor activity in GBM by multiple mechanisms.
  
  
  CONCLUSIONS
  The findings described here provide further evidence of oncogenic roles for MERTK in GBM, demonstrate the importance of kinase activity for MERTK tumorigenicity and validate UNC2025, a novel MERTK inhibitor, as a potential therapeutic agent for treatment of GBM.
authors: University of Colorado Anschutz Medical Campus, 12800 E 19th Avenue, Aurora, CO, 80045, United States of America.
authors: Alexandra
authors: A
authors: Sufit
authors: University of Colorado Anschutz Medical Campus, 12800 E 19th Avenue, Aurora, CO, 80045, United States of America.|Children's Hospital Colorado, 13123 E. 16th Ave, Aurora, CO, 80045, United States of America.
authors: Alisa B
authors: AB
authors: Lee-Sherick
authors: Aflac Cancer and Blood Disorders Center, Children's Healthcare of Atlanta and Emory University Department of Pediatrics, Atlanta, GA, 30322, United States of America.
authors: Deborah
authors: D
authors: DeRyckere
authors: Winship Cancer Institute, Emory University, Atlanta, GA, 30333, United States of America.
authors: Manali
authors: M
authors: Rupji
authors: Winship Cancer Institute, Emory University, Atlanta, GA, 30333, United States of America.
authors: Bhakti
authors: B
authors: Dwivedi
authors: University of Colorado Anschutz Medical Campus, 12800 E 19th Avenue, Aurora, CO, 80045, United States of America.
authors: Marileila
authors: M
authors: Varella-Garcia
authors: University of Colorado Anschutz Medical Campus, 12800 E 19th Avenue, Aurora, CO, 80045, United States of America.
authors: Angela M
authors: AM
authors: Pierce
authors: Winship Cancer Institute, Emory University, Atlanta, GA, 30333, United States of America.|Department of Biostatistics and Bioinformatics, Rollins School of Public Health, Emory University, Atlanta, GA, 30333, United States of America.
authors: Jeanne
authors: J
authors: Kowalski
authors: Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, United States of America.
authors: Xiaodong
authors: X
authors: Wang
authors: Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, United States of America.|Lineberger Comprehensive Cancer Center, Department of Medicine, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, Unites States of America.
authors: Stephen V
authors: SV
authors: Frye
authors: Lineberger Comprehensive Cancer Center, Department of Medicine, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, Unites States of America.
authors: H Shelton
authors: HS
authors: Earp
authors: University of Colorado Anschutz Medical Campus, 12800 E 19th Avenue, Aurora, CO, 80045, United States of America.|Children's Hospital Colorado, 13123 E. 16th Ave, Aurora, CO, 80045, United States of America.
authors: Amy K
authors: AK
authors: Keating
authors: Aflac Cancer and Blood Disorders Center, Children's Healthcare of Atlanta and Emory University Department of Pediatrics, Atlanta, GA, 30322, United States of America.|Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, United States of America.
authors: Douglas K
authors: DK
authors: Graham
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D016922:Cellular Senescence / Q000187:drug effects*; D004789:Enzyme Activation / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D005909:Glioblastoma / Q000473:pathology*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D058953:Neural Stem Cells / Q000187:drug effects / Q000473:pathology; D010879:Piperazines / Q000494:pharmacology*; D011123:Polyploidy; D011379:Prognosis; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors*; D020794:Receptor Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D000076202:c-Mer Tyrosine Kinase; D000099255:Growth Arrest-Specific Protein 6
chemical_list: D000225:Adenine; D000970:Antineoplastic Agents; D036341:Intercellular Signaling Peptides and Proteins; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D020794:Receptor Protein-Tyrosine Kinases; D000076202:c-Mer Tyrosine Kinase; D000099255:Growth Arrest-Specific Protein 6; C000597431:UNC2025; C097072:MERTK protein, human