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Suppl. Figure S1 shows "Initial MAPKi screen using metabolic activity as readout". Suppl. Figure S2 shows "Western blot validation of MAPK pathway alterations in HEK293T cells". Suppl. Figure S3 shows "Dose-response curves of MAPKi combination treatment in DKFZ-BT66 pDIPZ-CMV cells". Suppl. Figure S4 shows "Dose response curves of MAPKi combination treatment in BT 40 pDIPZ CMV cells". Suppl. Figure S5 shows "Isobolograms of MAPKi combination treatment in DKFZ-BT66 pDIPZ-CMV and BT-40 pDIPZ-CMV cells"

Includes the legends for Suppl. Table S1-S5.

Suppl. Table S4 shows "MAPKi combinations chosen based on low estimated IC50s and pharmaceutical company in consideration of future clinical studies".

IPA upstream regulators

Supplemental Table Legend

Figure S1: Markers of OIS in murine PA model Figure S2: Viability of DKFZ-BT66 cells under rIL1B treatment Figure S3: SASP factor protein levels in primary PAs Figure S4: Treatments of astrocytes with senolytic agents and combinations treatments of ABT-737 in DKFZ-BT66 cells

Background The sensitivity of myelocytomatosis oncogene (MYC) amplified medulloblastoma to class I histone deacetylase (HDAC) inhibition has been shown previously; however, understanding the underlying molecular mechanism is crucial for selection of effective HDAC inhibitors for clinical use. The aim of this study was to investigate the direct molecular interaction of MYC and class I HDAC2, and the impact of class I HDAC inhibition on MYC function. Methods Co-immunoprecipitation and mass spectrometry were used to determine the co-localization of MYC and HDAC2. Chromatin immunoprecipitation (ChIP) sequencing and gene expression profiling were used to analyze the co-localization of MYC and HDAC2 on DNA and the impact on transcriptional activity in primary tumors and a MYC amplified cell line treated with the class I HDAC inhibitor entinostat. The effect on MYC was investigated by quantitative real-time PCR, western blot, and immunofluorescence. Results HDAC2 is a cofactor of MYC in MYC amplified medulloblastoma. The MYC-HDAC2 complex is bound to genes defining the MYC-dependent transcriptional profile. Class I HDAC inhibition leads to stabilization and reduced DNA binding of MYC protein, inducing a downregulation of MYC activated genes (MAGs) and upregulation of MYC repressed genes (MRGs). MAGs and MRGs are characterized by opposing biological functions and by distinct enhancer-box distribution. Conclusions Our data elucidate the molecular interaction of MYC and HDAC2 and support a model in which inhibition of class I HDACs directly targets MYC’s transactivating and transrepressing functions.

Pilocytic astrocytomas as well as other pediatric low-grade gliomas (pLGG) exhibit genetic events leading to aberrant activation of the MAPK pathway. The most common alterations are KIAA1549:BRAF fusions and BRAFV600E and NF1 mutations. Novel drugs targeting the MAPK pathway (MAPKi) are prime candidates for the treatment of these single-pathway diseases. We aimed to develop an assay suitable for preclinical testing of MAPKi in pLGGs with the goal to identify novel MAPK pathway–suppressing synergistic drug combinations. A reporter plasmid (pDIPZ) with a MAPK-responsive ELK-1–binding element driving the expression of destabilized firefly luciferase was generated and packaged using a lentiviral vector system. Pediatric glioma cell lines with a BRAF fusion (DKFZ-BT66) and a BRAFV600E mutation (BT-40) background, respectively, were stably transfected. Modulation of the MAPK pathway activity by MAPKi was measured using the luciferase reporter and validated by detection of phosphorylated protein levels. A screening of a MAPKi library was performed, and synergy of selected combinations was calculated. Screening of a MAPKi library revealed MEK inhibitors as the class inhibiting the pathway with the lowest IC50s, followed by ERK and next-generation RAF inhibitors. Combination treatments with different MAPKi classes showed synergistic effects in BRAF fusion as well as BRAFV600E mutation backgrounds. Here, we report a novel reporter assay for medium- to high-throughput preclinical drug testing in pLGG cell lines. The assay confirmed MEK, ERK, and next-generation RAF inhibitors as potential treatment approaches for KIAA1549:BRAF and BRAFV600E-mutated pLGGs. In addition, the assay revealed that combination treatments synergistically suppressed MAPK pathway activity.

Pediatric low-grade glioma (pLGG), the most common brain tumors in children, are driven by alterations in the MAPK pathway. Several clinical trials have shown the potential for MAPK inhibitor (MAPKi) treatment in pLGG. However, the range of response to MAPKi is heterogeneous, even between tumors sharing the same driving MAPK alteration. A predictive stratification tool is needed to identify tumors that will be sensitive to MAPK inhibition. We generated sensitivity gene signatures for each MAPKi class (BRAFi, MEKi, ERKi), based on MAPK-related genes differentially regulated between MAPKi sensitive and non-sensitive cell lines from the Genomics of Drug Sensitivity in Cancer (GDSC) dataset. Single sample Gene Set Enrichment Analysis was used to measure and validate the MAPKi predictive sensitivity scores in the GDSC dataset and an independent patient-derived xenograft (PDX) dataset (XevaDB). The validated signatures were tested in a pLGG-specific background, using gene expression data from pLGG cell lines and primary pLGG samples. Our MAPKi sensitivity signatures discriminated MAPKi sensitive and non-sensitive cells in the GDSC dataset, and significantly correlated with MAPKi response in the PDX dataset. The sensitivity scores discerned gliomas with varying MAPK alterations from those without MAPK alterations, and showed higher scores in pLGG compared to high-grade gliomas and normal brain tissue. MAPKi-predicted sensitivity was heterogeneous within pLGG groups with a common MAPK alteration, as observed in MAPKi clinical trials. Intriguingly, we observed a strong positive correlation between our MAPKi sensitivity signature scores and the predicted immune cell infiltration rate as determined by the ESTIMATE score. These data demonstrate the potential relevance of gene-expression signatures to predict response to MAPKi treatment in pLGG patients, worth of further investigation in a prospective manner in upcoming clinical trials. In addition, our data could support a role of immune cell infiltration in the response to MAPKi in pLGG, warranting further validation.

Purpose: Pilocytic astrocytoma is the most common childhood brain tumor, characterized by constitutive MAPK activation. MAPK signaling induces oncogene-induced senescence (OIS), which may cause unpredictable growth behavior of pilocytic astrocytomas. The senescence-associated secretory phenotype (SASP) has been shown to regulate OIS, but its role in pilocytic astrocytoma remains unknown. Experimental Design: The patient-derived pilocytic astrocytoma cell culture model, DKFZ-BT66, was used to demonstrate presence of the SASP and analyze its impact on OIS in pilocytic astrocytoma. The model allows for doxycycline-inducible switching between proliferation and OIS. Both states were studied using gene expression profiling (GEP), Western blot, ELISA, and cell viability testing. Primary pilocytic astrocytoma tumors were analyzed by GEP and multiplex assay. Results: SASP factors were upregulated in primary human and murine pilocytic astrocytoma and during OIS in DKFZ-BT66 cells. Conditioned medium induced growth arrest of proliferating pilocytic astrocytoma cells. The SASP factors IL1B and IL6 were upregulated in primary pilocytic astrocytoma, and both pathways were regulated during OIS in DKFZ-BT66. Stimulation with rIL1B but not rIL6 reduced growth of DKFZ-BT66 cells and induced the SASP. Anti-inflammatory treatment with dexamethasone induced regrowth of senescent cells and inhibited the SASP. Senescent DKFZ-BT66 cells responded to senolytic BCL2 inhibitors. High IL1B and SASP expression in pilocytic astrocytoma tumors was associated with favorable progression-free survival. Conclusions: We provide evidence for the SASP regulating OIS in pediatric pilocytic astrocytoma, with IL1B as a relevant mediator. SASP expression could enable prediction of progression in patients with pilocytic astrocytoma. Further investigation of the SASP driving the unpredictable growth of pilocytic astrocytomas, and its possible therapeutic application, is warranted.

Pilocytic astrocytomas (PA) are the most common pediatric brain tumors. They are characterized by driving alterations in the mitogen-activated protein kinase (MAPK) pathway, leading to its constitutive activation and modulating the balance between cell proliferation and oncogene-induced senescence (OIS) sustained by senescence-associated secretory phenotype (SASP) factors. This makes PA susceptible to MAPK inhibitor (MAPKi) therapies, which show encouraging results in phase 1/2 clinical trials. However, little is known about the molecular implications of MAPK inhibition in PA. The DKFZ-BT66 cell line, derived from a primary KIAA:BRAF-fusion positive PA, was used as a model system. DKFZ-BT66 were treated with the MEKi trametinib for different durations in both proliferative and senescent states. Gene expression was analyzed by gene expression profiling and protein expression/phospho-regulation by data-dependent mass spectrometry followed by label-free quantitative analysis. A time course analysis based on differentially expressed genes and phosphorylated proteins was performed, followed by a single-sample gene set enrichment analysis (ssGSEA) and kinase substrate enrichment analysis, respectively. Differential gene expression analysis revealed that MEK inhibition led to the inhibition of the OIS/SASP gene programs in senescent DKFZ-BT66, with downregulation of key OIS/SASP partners such as IL1B on the protein level. This functionally translated into a de-sensitization of these cells towards the senolytic agent navitoclax. ssGSEA showed that most MAPK-related signatures were downregulated upon MEKi treatment, while pathways related to upstream MAPK activators (including FGFR, NTRK and TGFB pathways) were upregulated, in both proliferating and senescent DKFZ-BT66. This data indicates that MAPKi reverses OIS in senescent PA cells, while inducing the activation of MAPK upstream regulators in proliferating and senescent PA cells, identifying putative co-targets that could help increase treatment’s efficacy. Validation of these targets by post-translational modification enrichment analysis of the phospho-proteomics dataset is ongoing.

Introduction Pilocytic astrocytomas (PA) are the most common pediatric brain tumors. They are characterized by MAPK pathway alterations, leading to its constitutive activation and modulating the balance between cell proliferation and the oncogene-induced senescence (OIS) sustained by senescence-associated secretory phenotype (SASP) factors. This makes PA suitable for MAPK inhibitor (MAPKi) therapies, showing encouraging results in phase 1/2 clinical trials. Little is known about the molecular implications of MAPK downregulation in the proliferating and senescent compartments. Methods DKFZ-BT66 PA cells derived from a primary KIAA:BRAF-fusion positive PA cell line, were used as model system. Gene expression and phospho-proteomic datasets were generated from DKFZ-BT66 cells, in both the proliferative and senescent states, and treated with the MEKi trametinib for different time-spans. A time course analysis based on differentially expressed genes was performed, followed by a single-sample gene set enrichment analysis (ssGSEA). Analysis of the phospho-proteomic data is ongoing. Results Differential gene expression analysis revealed that MEK inhibition leads to the inhibition of the OIS-SASP gene program in senescent DKFZ-BT66. ssGSEA showed that most MAPK-related signatures were downregulated upon MEKi treatment, while pathways related to upstream MAPK activators (including FGFR, NTRK and TGFB pathways) were upregulated, in both proliferating and senescent DKFZ-BT66. Genes regulated by the MAPK pathway and involved in OIS-SASP were identified by analyzing genes differentially regulated between proliferating and senescent DKFZ-BT66, and modulated upon MEKi treatment. Conclusion This data suggests that MAPKi reverses OIS in senescent PA cells, while inducing the activation of MAPK upstream regulators in proliferating and senescent PA cells, identifying putative co-targets that could help prevent growth rebound upon MAPKi withdrawal. Furthermore, the identification of the MAPK-related OIS-SASP genes provide insight about the regulation of OIS-SASP by the MAPK pathway. Validation of this data with the ongoing phospho-proteomic analysis and in primary samples is needed.

Pilocytic astrocytomas as well as other pediatric low-grade gliomas (pLGG) exhibit genetic events leading to aberrant activation of the MAPK pathway. The most common alterations are

// Florian Selt 1, 2 , Juliane Hohloch 1 , Thomas Hielscher 3 , Felix Sahm 4, 5 , David Capper 4, 5 , Andrey Korshunov 4, 5 Diren Usta 1 , Sebastian Brabetz 6 , Johannes Ridinger 1 , Jonas Ecker 1, 2 , Ina Oehme 1 , Jan Gronych 7, 8 , Viktoria Marquardt 9 , David Pauck 9 , Heidi Bachli 10 , Charles D. Stiles 11 , Andreas von Deimling 4, 5 , Marc Remke 9 , Martin U. Schuhmann 12 , Stefan M. Pfister 2, 6 , Tilman Brummer 13 , David T.W. Jones 6 , Olaf Witt 1, 2, * , Till Milde 1, 2, * 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, and German Cancer Consortium (DKTK), Heidelberg, Germany 7 Division of Molecular Genetics (B060), German Cancer Research Center (DKFZ), and German Cancer Consortium (DKTK), Heidelberg, Germany 8 AbbVie Deutschland GmbH & Co. KG, Medical Immunology, Wiesbaden, Germany (current affiliation) 9 Department of Pediatric Oncology, Hematology, and Clinical Immunology, Medical Faculty, University Hospital Dusseldorf, Germany, and Department of Pediatric Neuro-Oncogenomics, German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany 10 Department of Neurosurgery, University Hospital Heidelberg, Heidelberg, Germany 11 Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA 12 Department of Neurosurgery, University Hospital Tubingen, Tubingen, Germany 13 Institute 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. selt@dkfz.de 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.

Pilocytic astrocytomas (PA) are low-grade gliomas (pLGG) and are the most frequent childhood brain tumors. They are characterized by oncogene-induced senescence (OIS) initiated and sustained by senescence-associated secretory phenotype (SASP) factors. OIS and SASP in PA are thought to be driven by aberrations of the mitogen-activated protein kinase (MAPK) pathway (e.g. KIAA1549:BRAF fusion, BRAFV600E mutation, for the most common MAPK alterations occuring in PA), leading to its sustained activation. The MAPK pathway cascade is activated in a sequential manner: 1) ERK activation, which phosphorylates downstream partners in both cytoplasm and nucleus. 2) ERK-mediated induction of immediate early genes encoding transcription factors. 3) Induction of MAPK target genes expression. 4) Activation of downstream pathways. Our aim is to unravel the molecular partners involved at each level of the sustained MAPK pathway activation in pLGG with different genetic backgrounds (KIAA1549:BRAF fusion and BRAFV600E mutation), and leading to the induction of OIS and SASP factors expression. pLGG cell lines DKFZ-BT66 (KIAA1549:BRAF) and BT-40 (BRAFV600E) were treated with the MEK inhibitor trametinib at key time points, and gene expression profile analysis was performed, allowing transcriptome analysis at each step of the MAPK cascade. This will be combined with a whole proteomic and phospho-proteomic analysis. Combination of the transcriptome and proteome data layers will allow the identification of a) downstream targetable partners activated by the MAPK pathway involved in PA senescence, b) new putative targets that might bring benefit in combination with MAPK inhibitors.

Pilocytic astrocytomas (PAs) and other pediatric low-grade gliomas (pLGGs) exhibit aberrant activation of the MAPK signaling pathway caused by genetic alterations, most commonly KIAA1549:BRAF fusions, BRAF V600E and NF1 mutations. In such a single-pathway disease, novel drugs targeting the MAPK pathway (MAPKi) are prime candidates for treatment. We developed an assay suitable for pre-clinical testing of MAPKi in pLGGs, aiming at the identification of novel MAPK pathway suppressing synergistic drug combinations. We generated a reporter plasmid (pDIPZ) expressing destabilized firefly luciferase driven by a MAPK-responsive ELK-1-binding element, packaged in a lentiviral vector system. We stably transfected pediatric glioma cell lines with a BRAF fusion (DKFZ-BT66) and a BRAFV600E mutation (BT-40) background, respectively. Measurement of MAPK pathway activity was performed using the luciferase reporter. pERK protein levels were detected for validation. We performed a screen of a MAPKi library and calculated Combination Indices of selected combinations. The MAPKi library screen revealed MEK inhibitors as the class inhibiting the pathway with the lowest IC50s, followed by ERK and second generation RAF inhibitors. Synergistic effects in both BRAF-fusion and BRAFV600E mutation backgrounds were observed following combination treatments with different MAPKi classes (RAFi/MEKi, > RAFi/ERKi > MEKi/ERKi). We have generated a novel reporter assay for medium- to high-throughput pre-clinical drug testing of MAPKi in pLGG cell lines. MEK, ERK and next-generation RAF inhibitors were confirmed as potential treatment approaches for KIAA1549:BRAF and BRAFV600E mutated pLGGs. Synergistic suppression of MAPK pathway activity upon combination treatments was revealed using our assay in addition.

High-risk medulloblastoma (MB) is a deadly disease with poor overall survival. Survivors suffer from severe treatment-associated morbidity. Patients with Group 3 MB with an amplification of MYC show particularly poor outcome. Therefore novel therapies tailored to this subgroup are urgently needed. We have previously shown that MYC amplified MB cell lines are highly susceptible to inhibition of class I histone deacetylases (HDACs), including HDAC2. We here explore the functional interaction of HDAC2 and MYC. METHODS: Chromatin immunoprecipitation with pulldown of MYC, HDAC2, RNApolII and H3K27ac, followed by DNA-sequencing was performed in n=3 primary MYC amplified Group 3 MB tumors and correlated with gene expression profiles (GEP). The GEP of MYC amplified MB cell line HD-MB03 was determined after class I HDAC inhibition. MYC stability upon HDACi treatment was assessed in immunofluorescence microscopy and cycloheximide chase experiments. MYC-HDAC2 interaction was analyzed in co-immunoprecipitation and mass spectrometry studies. RESULTS: In primary tumors 90% of MYC DNA binding sites are co-occupied by HDAC2. Compared to MYC- or HDAC2-only bound genes, MYC-HDAC2 co-bound genes show strong recruitment of RNApolII and higher expression levels. In HD-MB03 cells MYC and HDAC2 show overlapping DNA binding sites of 80%, both proteins are co-localized in a multi-protein complex. GEP analysis revealed a significant downregulation of MYC target genes after treatment with class I HDAC inhibitor MS-275, coinciding aberrant MYC protein levels. Our data suggests that MYC functions directly depend on class I HDAC activity. Inhibiting HDAC2 may offer a therapeutic option to target the currently undruggable oncoprotein MYC.

Pilocytic astrocytoma (PA) is the most frequent pediatric brain tumor. This single-pathway disease exhibits aberrant activation of the MAPK pathway driving the tumor into oncogene-induced senescence (OIS). OIS is proposed to be the source of the unstable however benign growth observed in PA patients. Senescence is thought to be regulated by the senescence-associated secretory phenotype (SASP) which comprises a variety of cytokines, growth factors and proteases. Markers of senescence have been detected in PA, but the functional relevance of the SASP and its relation to OIS in PA is unknown. The first patient-derived PA cell culture model DKFZ-BT66 was utilized for the characterization of OIS and the role of the SASP in PA. The model allows experimental switching between senescent and proliferating states by modulation of the p53/RB pathway. Both conditions were analyzed by gene-expression profiling (GEP), Western Blot, real-time qPCR, ELISA, cell counts and viability by automated trypan blue exclusion staining. A significant increase of the SASP could be detected by GEP in the OIS state of the PA cell line. Moreover, the OIS expression signature was associated with improved progression free survival in a cohort of n=112 PA patients. Upregulation of IL-6 and IL-1B, two representative SASP factors, could be demonstrated on mRNA and protein level in DKFZ-BT66 during OIS. Both cytokine receptors are expressed and activation of the respective pathways was confirmed. Activation of the IL-1 pathway led to decreased growth of proliferating PA cells. Overall, the novel primary PA tumor model provides functional evidence of the presence of OIS in PA and exhibits increased activity of the SASP during the senescent state. In order to find an explanation for the clinically observed spontaneous on/off growth behavior, current studies aim to investigate the disruption of the OIS-characteristic growth arrest by inhibition of inflammatory signaling pathways.

The main challenge in the clinical management of pilocytic astrocytoma (PA) is its unpredictable growth behavior. PA cells are driven into oncogene-induced senescence (OIS) by aberrant MAPK activation. In other senescence models OIS was shown to be regulated and maintained by the senescence-associated secretory phenotype (SASP). In this study, the first patient-derived cell culture model DKFZ-BT66 was used to show presence of the SASP in PA and to analyze its impact on OIS. This model allows for shifting between proliferation and senescence via doxycycline-inducible inhibition of the OIS-relevant p53/RB pathway. Both states were studied using gene-expression profiling (GEP), Western blot, qPCR, ELISA, and automated trypan blue exclusion staining. The GEP shows significant upregulation of SASP factors during OIS in DKFZ-BT66 cells. Conditioned medium of senescent DKFZ-BT66 cells is sufficient to induce growth arrest of proliferating DKFZ-BT66 cells. Upregulation of the SASP factors IL-1B and IL-6 was validated on mRNA and protein levels and both pathways are active during OIS. Stimulation of the IL-1 pathway reduces growth of proliferating DKFZ-BT66 cells. While pharmacological inhibition of single cytokines is not sufficient to overcome growth arrest during OIS, treatment with the anti-inflammatory drug dexamethasone induces regrowth of senescent cells. Overall, the primary PA model provides evidence of the presence of OIS in PA and exhibits increased activity of the SASP during senescence. Our data suggest that the SASP has an important impact on the growth regulation of senescent PA cells. Alteration of SASP factors may result in spontaneous regrowth of senescent PA tumors.

The benign but unpredictable growth behaviour of pilocytic astrocytoma (PA) is thought to originate from variations in proliferation caused by oncogene-induced senescence (OIS). An increase in MAPK pathway activation, present in virtually all PAs, serves a stimulus driving the tumor into OIS. Upregulation of p16, p21 and several other OIS-related genes has been observed in primary PAs. The secretion of a set of cytokines, growth factors and proteases, referred to as the senescence-associated secretory phenotype (SASP), is a characteristic feature of senescent cells. The SASP is thought to be involved in the induction and maintenance of the OIS state. The key SASP factors in PA and their functional significance are unknown. To study OIS in PA, the genetically engineered primary PA cell culture model DKFZ-BT66 was used. The model allows switching between senescence and proliferation via inducible expression of the SV40 Large T Antigen suppressing p53/RB signaling. Readouts used were gene-expression profiling (GEP), Western Blot, real-time qPCR, ELISA, cell counts and viability by automated trypan blue exclusion staining. Comparison of the GEP of senescent versus proliferating cells showed a significant increase in the SASP during OIS. The upregulation of the representative SASP factors IL-6 and IL-1B was confirmed on mRNA and protein level. The respective receptor proteins were both expressed, and activation of both pathways was detected in the OIS state. Stimulation of selected SASP pathways led to reduced cell growth of proliferating cells. In summary, the first representative patient-derived PA tumor model DKFZ-BT66 confirms the presence of OIS in PA, and additionally reveals activity of the OIS-characteristic SASP. Ongoing studies will show whether the OIS-induced growth arrest is reversible and if it may explain the unpredictable regrowth of PAs observed in patients. These findings will be relevant for treatment decisions concerning anti-inflammatory or immunosuppressive drugs in PA patients.