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Abstract

Brain tumors are the second most common malignancy diagnosed in children, with low-grade gliomas (LGG) being the most common childhood brain tumor, and pilocytic astrocytoma (PA) the most common LGG. LGGs are typically driven by aberrant MAPK pathway activation commonly induced by BRAF fusions or mutations. These genetic alterations activate the tumor-suppressive mechanism oncogene-induced senescence (OIS), resulting in growth arrest of transformed cells. OIS has been shown to be regulated by a complex network of inflammatory molecules, referred to as the senescence-associated secretory phenotype (SASP). Single markers of OIS have been detected in primary PAs, but its functional role in PA remains unknown to date. A patient-derived PA cell line with a BRAF fusion was generated via lentiviral transduction with a doxycycline-inducible construct coding for the SV40 Large T antigen (SV40-TAg). This novel PA model, DKFZ-BT66, enabled the analysis of the growth-arrested OIS state of PA cells as well as the proliferating state during SV40-TAg expression. Both conditions were characterized and analyzed by means of gene expression profiling (GEP), western blot, ELISA and cell viability testing via automated trypan blue exclusion staining. Primary PA material was analyzed by GEP as well as a multiplex assay. The SASP was upregulated in the OIS state of the human PA cell line DKFZ-BT66 as well as in primary human and murine PAs. Conditioned medium of senescent cells was shown to arrest growth and induce the senescence-characteristic enlarged cellular phenotype in proliferating PA cells. The SASP factors IL1B and IL6 were both upregulated and secreted by senescent PA cells and their respective pathways were shown to be regulated during OIS. Treatment of proliferating DKFZ-BT66 cells with recombinant IL1B (rIL1B), but not rIL6, reduced cell growth of proliferating PA cells. Both SASP expression as well as changes in cell morphology, reminiscent of the enlarged senescent phenotype, were induced by rIL1B treatment. However, neither pharmacological nor shRNA-mediated inhibition of the IL1 or IL6 pathway led to a bypass of the OIS state in the DKFZ-BT66 cell line. Treatment with the anti-inflammatory drug dexamethasone induced regrowth of senescent DKFZ-BT66 cells and suppressed SASP gene expression. The clinical relevance of the SASP in PA was confirmed by the identification of two patient cohorts with differing clinical outcome related to SASP expression. Elevated expression of the SASP as well as of IL1B alone was predictive for favorable progression-free survival (PFS) in PA patients independent of tumor resection status. To exploit OIS therapeutically, DKFZ-BT66 cells were treated with senolytic BCL2 family member inhibitors, specifically targeting senescent cells. Senescent PA cells were more sensitive to senolytics in comparison to proliferating DKFZ-BT66 cells or normal human astrocytes. In summary, the SASP was shown to regulate OIS in pediatric PA, with IL1B as an important mediator. Elevated SASP expression was prognostic for a favorable PFS in the analyzed cohort and will have to be validated as a prognostic marker in prospective clinical trials. The combination of senolytic agents, targeting senescent PA cells, together with chemotherapy, targeting cycling PA cells, may be a novel therapeutic approach and will have to be evaluated in further preclinical studies.