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@ARTICLE{Selt:124405,
      author       = {F. Selt$^*$ and J. Hohloch$^*$ and T. Hielscher$^*$ and F.
                      Sahm$^*$ and D. Capper$^*$ and A. Korshunov$^*$ and D.
                      Usta$^*$ and S. Brabetz$^*$ and J. Ridinger$^*$ and J.
                      Ecker$^*$ and I. Oehme$^*$ and J. Gronych$^*$ and V.
                      Marquardt$^*$ and D. Pauck$^*$ and H. Bächli and C. D.
                      Stiles and A. von Deimling$^*$ and M. Remke$^*$ and M. U.
                      Schuhmann and S. Pfister$^*$ and T. Brummer and D. Jones$^*$
                      and O. Witt$^*$ and T. Milde$^*$},
      title        = {{E}stablishment and application of a novel patient-derived
                      {KIAA}1549:{BRAF}-driven pediatric pilocytic astrocytoma
                      model for preclinical drug testing.},
      journal      = {OncoTarget},
      volume       = {8},
      number       = {7},
      issn         = {1949-2553},
      address      = {[S.l.]},
      publisher    = {Impact Journals LLC},
      reportid     = {DKFZ-2017-01284},
      pages        = {11460-11479},
      year         = {2017},
      abstract     = {Pilocytic astrocytoma (PA) is the most frequent pediatric
                      brain tumor. Activation of the MAPK pathway is well
                      established as the oncogenic driver of the disease. It is
                      most frequently caused by KIAA1549:BRAF fusions, and leads
                      to oncogene induced senescence (OIS). OIS is thought to be a
                      major reason for growth arrest of PA cells in vitro and in
                      vivo, preventing establishment of PA cultures. Hence, valid
                      preclinical models are currently very limited, but
                      preclinical testing of new compounds is urgently needed. We
                      transduced the PA short-term culture DKFZ-BT66 derived from
                      the PA of a 2-year old patient with a doxycycline-inducible
                      system coding for Simian Vacuolating Virus 40 Large T
                      Antigen (SV40-TAg). SV40-TAg inhibits TP53/CDKN1A and
                      CDKN2A/RB1, two pathways critical for OIS induction and
                      maintenance. DNA methylation array and KIAA1549:BRAF fusion
                      analysis confirmed pilocytic astrocytoma identity of
                      DKFZ-BT66 cells after establishment. Readouts were analyzed
                      in proliferating as well as senescent states, including cell
                      counts, viability, cell cycle analysis, expression of
                      SV40-Tag, CDKN2A (p16), CDKN1A (p21), and TP53 (p53)
                      protein, and gene-expression profiling. Selected MAPK
                      inhibitors (MAPKi) including clinically available MEK
                      inhibitors (MEKi) were tested in vitro. Expression of
                      SV40-TAg enabled the cells to bypass OIS and to resume
                      proliferation with a mean doubling time of 45h allowing for
                      propagation and long-term culture. Withdrawal of doxycycline
                      led to an immediate decrease of SV40-TAg expression,
                      appearance of senescent morphology, upregulation of CDKI
                      proteins and a subsequent G1 growth arrest in line with the
                      re-induction of senescence. DKFZ-BT66 cells still underwent
                      replicative senescence that was overcome by TERT expression.
                      Testing of a set of MAPKi revealed differential responses in
                      DKFZ-BT66. MEKi efficiently inhibited MAPK signaling at
                      clinically achievable concentrations, while BRAF V600E- and
                      RAF Type II inhibitors showed paradoxical activation. Taken
                      together, we have established the first patient-derived long
                      term expandable PA cell line expressing the
                      KIAA1549:BRAF-fusion suitable for preclinical drug testing.},
      cin          = {G340 / C060 / G380 / B062 / B060 / L401 / L101 / L601},
      ddc          = {610},
      cid          = {I:(DE-He78)G340-20160331 / I:(DE-He78)C060-20160331 /
                      I:(DE-He78)G380-20160331 / I:(DE-He78)B062-20160331 /
                      I:(DE-He78)B060-20160331 / I:(DE-He78)L401-20160331 /
                      I:(DE-He78)L101-20160331 / I:(DE-He78)L601-20160331},
      pnm          = {317 - Translational cancer research (POF3-317)},
      pid          = {G:(DE-HGF)POF3-317},
      typ          = {PUB:(DE-HGF)16},
      pubmed       = {pmid:28002790},
      pmc          = {pmc:PMC5355278},
      doi          = {10.18632/oncotarget.14004},
      url          = {https://inrepo02.dkfz.de/record/124405},
}