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Oncotarget. 2017 Feb 14;8(7):11460-11479. doi: 10.18632/oncotarget.14004.

Establishment and application of a novel patient-derived KIAA1549:BRAF-driven pediatric pilocytic astrocytoma model for preclinical drug testing.

Author information

1
Clinical Cooperation Unit Pediatric Oncology (G340), German Cancer Research Center (DKFZ), and German Cancer Consortium (DKTK), Heidelberg, Germany.
2
Center for Individualized Pediatric Oncology (ZIPO) and Section of Pediatric Brain Tumors, Department of Pediatric Oncology, Hematology and Immunology, University Hospital Heidelberg, Heidelberg, Germany.
3
Division of Biostatistics (C060), German Cancer Research Center (DKFZ), Heidelberg, Germany.
4
Department of Neuropathology, University Hospital Heidelberg, Heidelberg, Germany.
5
Clinical Cooperation Unit Neuropathology (G380), German Cancer Research Center (DKFZ), and German Cancer Consortium (DKTK), Heidelberg, Germany.
6
Division of Pediatric Neurooncology (B062), German Cancer Research Center (DKFZ), Heidelberg, Germany.
7
German Cancer Consortium (DKTK), Heidelberg, Germany.
8
Division of Molecular Genetics (B060), German Cancer Research Center (DKFZ), and German Cancer Consortium (DKTK), Heidelberg, Germany.
9
Current affiliation: AbbVie Deutschland GmbH & Co. KG, Medical Immunology, Wiesbaden, Germany.
10
Department of Pediatric Oncology, Hematology, and Clinical Immunology, Medical Faculty, University Hospital Düsseldorf, Germany.
11
Department of Pediatric Neuro-Oncogenomics, German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany.
12
Department of Neurosurgery, University Hospital Heidelberg, Heidelberg, Germany.
13
Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA.
14
Department of Neurosurgery, University Hospital Tübingen, Tübingen, Germany.
15
Institute of Molecular Medicine and Cell Research, Albert-Ludwigs-University and University Medical Centre, Freiburg, Germany.
16
Division of Pediatric Neurooncology (B062), German Cancer Research Center (DKFZ), Heidelberg, Germany, and German Cancer Consortium (DKTK), Heidelberg, Germany.

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.

KEYWORDS:

KIAA1549:BRAF-fusion; MAPK-inhibitors; oncogene-induced senescence (OIS); pediatric low grade glioma; pilocytic astrocytoma

PMID:
28002790
PMCID:
PMC5355278
DOI:
10.18632/oncotarget.14004
[Indexed for MEDLINE]
Free PMC Article

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