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

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.

The miR‐139‐5p regulates proliferation of supratentorial paediatric low‐grade gliomas by targeting the PI3K/AKT/mTORC1 signalling

“…In DNET and GG samples, we also detected frequent positivity for NeuN and Synaptophysin, neuronal markers, which conversely were absent in the AG. Therefore, the immunofluorescence staining together with the methylation profiling, a method to evaluate the similarity among tumours samples and their derived primary cells , indicate that our primary cells are a suitable and trustworthy model to study supratentorial pLGGs biology.…”

“…A major drawback of these cell lines is that they harbour genetic alterations that are relatively rare in pLGGs, and it is unclear to what extent they represent the primary lesions . Previous attempts to use short‐term primary cultures of patient‐derived pLGGs have been hampered by markedly reduced growth secondary to oncogene induced senescence . Protocol differences might contribute to explain why our efforts were more successful.…”

“…For instance, in some cultures, high rates were already observed after the first few passages. By carefully selecting the cultures destined for experiments particularly by analysing their whole genome methylation profile (Figure S4), demonstrated to be a reliable method to pinpoint the identity of a broad range of brain tumours , but also continuously monitoring their senescence rates over time, we were able to complete short‐term experiments and our assessment of these cells indicates that they are likely to provide reliable representations of the pLGGs seen in the clinic. Therefore, our primary patient‐derived pLGG cells present many benefits in comparison with the few immortalized cell lines that have been used until now and represent a more reliable model to study the biology of these tumours, that otherwise would be hardly addressed.…”

“…Other mutations, such as FGFR1 alterations, are mainly expressed only by specific pLGGs subtypes, such as DNET . Along with the dysregulated MAPK/ERK signalling mentioned above , aberrant activation of the PI3K/AKT signalling pathway, which can be demonstrated by high levels of phosphorylated AKT (p‐AKT), is also a well‐documented feature of gliomas, including pLGGs . In some cases, its dysregulation has been linked to genetic changes, such as BRAF fusions, FGFR1 duplications and MYB rearrangements , or epigenetic modifications, but in many cases the mechanism underlying PI3K/AKT pathway activation is still unknown.…”

CRAF gene fusions in pediatric low-grade gliomas define a distinct drug response based on dimerization profiles

“…Relevant patient-derived models are extremely rare for PLGGs, with no established PLGG cell lines expressing CRAF fusions. 47 , 48 , 49 The heterologous model systems used in this study have previously been predictive of clinical response such as in the PLGG Phase II trial with sorafenib. 29 These model systems indicate that CRAF fusions are oncogenic via the MAPK and PI3K/mTOR pathways.…”