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  • 1
    In: Neuro-Oncology, 2017, Vol. 19(suppl6), pp.vi200-vi200
    Description: 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.
    Keywords: Medicine;
    ISSN: 1522-8517
    E-ISSN: 1523-5866
    Source: Oxford University Press
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  • 2
    In: Neuro-Oncology, 2018, Vol. 20(suppl2), pp.i106-i106
    Description: 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.
    Keywords: Medicine;
    ISSN: 1522-8517
    E-ISSN: 1523-5866
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  • 3
    Language: English
    In: Oncotarget, 14 February 2017, Vol.8(7), pp.11460-11479
    Description: 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.
    Keywords: Kiaa1549:Braf-Fusion ; Mapk-Inhibitors ; Oncogene-Induced Senescence (Ois) ; Pediatric Low Grade Glioma ; Pilocytic Astrocytoma ; Astrocytoma ; Brain Neoplasms ; Cell Culture Techniques ; Cell Line, Tumor ; Cellular Senescence -- Physiology
    E-ISSN: 1949-2553
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  • 4
    Language: English
    In: Clinical cancer research : an official journal of the American Association for Cancer Research, 15 March 2019, Vol.25(6), pp.1851-1866
    Description: 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. 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. 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 and SASP expression in pilocytic astrocytoma tumors was associated with favorable progression-free survival. 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.
    ISSN: 1078-0432
    E-ISSN: 15573265
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  • 5
    Language: English
    In: Neuro-Oncology, 06/2017, 06/01/2017, Vol.19(suppl_4), pp.iv33-iv33
    Keywords: Medicine;
    ISSN: 1522-8517
    E-ISSN: 1523-5866
    Source: Oxford University Press (via CrossRef)
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