Preclinical model development for pilocytic astrocytoma

Pilocytic astrocytoma (PA), the most frequent pediatric brain tumor, exhibits an activation of MAPK-signaling by genetic aberration in nearly 100% of cases, with activating KIAA1549:BRAF-fusions being the most frequent alteration of this pathway (60 – 80%). Dealing with a single-pathway disease evokes hope for improvement of PA treatment by small molecule inhibitors of the MAPK-signalling pathway. However, faithful preclinical models are currently very limited. The paradoxical acceleration of tumor growth in PA patients treated with sorafenib, a multi-kinase inhibitor targeting the Raf pathway amongst others, highlights the need for preclinical modeling before going into clinical trials. Our previous attempts to grow over 60 samples of primary low grade tumors in vitro and in vivo were unsuccessful, in contrast to primary high grade pediatric brain tumors. Hence novel approaches are needed to generate models for preclinical testing. Oncogene induced senescence (OIS) is a tumor suppressive mechanism that prevents tumors from further malignant transformation by induction of cell cycle arrest. OIS can often be found in RAS and RAF driven tumors and was shown to be present in the vast majority of PA samples as detected by SA-β-Gal-positivity and strong up-regulation of CDKIs (p16, p21) compared to normal brain tissue. We therefore hypothesize that OIS is likely the reason for growth arrest of PA cells in vitro and we aim to establish PA models by bypassing the cellular senescence program. Simian Virus 40 Large T Antigen (SV40-LTAg) is able to interfere with two pathways critically involved in OIS, p53-p21 and p16/pRB. A doxycycline inducible lentiviral expression system coding for SV40-LTAg will be used to infect primary PA cells taken from short term culture and normal human astrocytes transduced with PA-typical genetic alterations (KIAA:BRAF-fusions, BRAF V600E mutation). The inducibility of SV40-LTAg expression will enable for temporary cell expansion while also allowing for removal of the artificial growth stimulus by withdrawal of doxycycline for further experiments, e.g. testing of MEK-inhibitors. Furthermore, this non-permanent growth stimulus will reduce the likelihood for malignant transformation of the PA cells.