Oncotarget

Research Papers:

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

Florian Selt _, Juliane Hohloch, Thomas Hielscher, Felix Sahm, David Capper, Andrey Korshunov, Diren Usta, Sebastian Brabetz, Johannes Ridinger, Jonas Ecker, Ina Oehme, Jan Gronych, Viktoria Marquardt, David Pauck, Heidi Bächli, Charles D. Stiles, Andreas von Deimling, Marc Remke, Martin U. Schuhmann, Stefan M. Pfister, Tilman Brummer, David T.W. Jones, Olaf Witt and Till Milde

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Oncotarget. 2017; 8:11460-11479. https://doi.org/10.18632/oncotarget.14004

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Abstract

Florian Selt1,2, Juliane Hohloch1, Thomas Hielscher3, Felix Sahm4,5, David Capper4,5, Andrey Korshunov4,5 Diren Usta1, Sebastian Brabetz6, Johannes Ridinger1, Jonas Ecker1,2, Ina Oehme1, Jan Gronych7,8, Viktoria Marquardt9, David Pauck9, Heidi Bächli10, Charles D. Stiles11, Andreas von Deimling4,5, Marc Remke9, Martin U. Schuhmann12, Stefan M. Pfister2,6, Tilman Brummer13, David T.W. Jones6, Olaf Witt1,2,*, Till Milde1,2,*

1Clinical Cooperation Unit Pediatric Oncology (G340), German Cancer Research Center (DKFZ), and German Cancer Consortium (DKTK), Heidelberg, Germany

2Center for Individualized Pediatric Oncology (ZIPO) and Section of Pediatric Brain Tumors, Department of Pediatric Oncology, Hematology and Immunology, University Hospital Heidelberg, Heidelberg, Germany

3Division of Biostatistics (C060), German Cancer Research Center (DKFZ), Heidelberg, Germany

4Department of Neuropathology, University Hospital Heidelberg, Heidelberg, Germany

5Clinical Cooperation Unit Neuropathology (G380), German Cancer Research Center (DKFZ), and German Cancer Consortium (DKTK), Heidelberg, Germany

6Division of Pediatric Neurooncology (B062), German Cancer Research Center (DKFZ), Heidelberg, Germany, and German Cancer Consortium (DKTK), Heidelberg, Germany

7Division of Molecular Genetics (B060), German Cancer Research Center (DKFZ), and German Cancer Consortium (DKTK), Heidelberg, Germany

8AbbVie Deutschland GmbH & Co. KG, Medical Immunology, Wiesbaden, Germany (current affiliation)

9Department of Pediatric Oncology, Hematology, and Clinical Immunology, Medical Faculty, University Hospital Düsseldorf, Germany, and Department of Pediatric Neuro-Oncogenomics, German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany

10Department of Neurosurgery, University Hospital Heidelberg, Heidelberg, Germany

11Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA

12Department of Neurosurgery, University Hospital Tübingen, Tübingen, Germany

13Institute of Molecular Medicine and Cell Research, Albert-Ludwigs-University and University Medical Centre, Freiburg, Germany

*These authors have contributed equally to this work

Correspondence to:

Florian Selt, email: f. [email protected]

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

Received: November 18, 2016     Accepted: November 23, 2016     Published: December 17, 2016

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.


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