The Senescence-associated Secretory Phenotype Mediates Oncogene-induced Senescence in Pediatric Pilocytic Astrocytoma

SASP factors are upregulated in primary pilocytic astrocytoma, during OIS in pilocytic astrocytoma cells and induce growth arrest

We analyzed mRNA expression of the SASP factors in human pilocytic astrocytoma patient samples (n = 182) and compared it with normal brain (n = 5) by means of gene expression microarrays. The SASP gene set, as defined by Coppé (17), was significantly upregulated in human pilocytic astrocytomas (Fig. 1A). Likewise, the SASP gene set was significantly upregulated in a murine in vivo pilocytic astrocytoma model expressing a BRAF^V600E mutation (12; n = 8) compared with healthy mice of the same age (n = 8; Fig. 1B). Using the same approach, we analyzed the regulation of the SASP gene set in OIS versus proliferation in our pilocytic astrocytoma cell line, DKFZ-BT66. The OIS state in DKFZ-BT66 was defined at five days after doxycycline withdrawal, coinciding with enlarged cell morphology, SV40 large T antigen protein degradation, cell-cycle arrest, and upregulation of markers of senescence such as CDKN1A as described previously (14). Comparison of the SASP gene set in OIS versus proliferation showed a significant upregulation of the SASP gene set overall (Fig. 1C), with 38 of 62 (61%) of the SASP factors significantly upregulated (Fig. 1D; Supplementary Table S3). To determine the secretory nature and the functional relevance of the SASP for the induction of OIS in pilocytic astrocytoma, conditioned medium (CM) experiments were conducted. CM of either senescent or proliferating DKFZ-BT66 cells was administered to proliferating DKFZ-BT66 cells together with doxycycline for continuous induction of SV40 large T antigen expression. While cells treated with CM from proliferating pilocytic astrocytoma cells continued to grow, treatment with CM from senescent cells induced growth arrest (Fig. 1E) as well as an enlarged cell morphology characteristic for senescence (ref. 11; Fig. 1F). Taken together, these data suggest that the SASP is upregulated in human and murine pilocytic astrocytoma as well as in our pilocytic astrocytoma cell line, is secreted during OIS in DKFZ-BT66 cells and is sufficient to induce growth arrest of proliferating cells.

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Figure 1.

Upregulated SASP factors in pediatric pilocytic astrocytoma cells induce growth arrest. A, Barcode plot of gene-set enrichment analysis (GSEA) depicting significant upregulation of the SASP genes in pilocytic astrocytoma (PA) patient samples (n = 182) versus fetal normal brain (n = 5). B, Barcode plot of GSEA depicting significant upregulation of the SASP genes in the murine pilocytic astrocytoma model (n = 8) versus normal brain of healthy mice of the same age (n = 8). C, Barcode plot of GSEA depicting significant upregulation of the SASP genes in DKFZ-BT66 cells in OIS versus proliferation. D, Volcano plot depicting all probe sets regulated in DKFZ-BT66 during OIS, as compared with proliferation. E, Cell count of proliferating DKFZ-BT66 cells treated with CM every second day from DKFZ-BT66 cells in proliferation (blue) or OIS (red), supplemented with doxycycline (1 μg/mL) for 20 days. Depicted are mean ± SD of three independent experiments. Significant differences are indicated as *, P < 0.05 (Student t test). F, Light microscopic comparison of DKFZ-BT66 cells grown under CM from proliferating or senescent (OIS) DKFZ-BT66 cells (as in E) at day 20.

Identification of the OIS-controlling candidate genes IL1B and IL6 in pediatric pilocytic astrocytoma

As the composition of SASP factors differs depending on tissue and cell type (16), we next investigated which SASP factors as well as other OIS-controlling genes are expressed in pediatric human pilocytic astrocytomas. To identify specific OIS-controlling genes for pediatric pilocytic astrocytoma, we collected and generated three candidate gene lists of relevant OIS and SASP genes (step 1), investigated the expression of these genes in primary human pilocytic astrocytoma samples (step 2), and generated a final candidate gene list after accounting for consensus in the three candidate gene lists as well as druggability (step 3; Fig. 2A).

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Figure 2.

Validation of OIS-controlling candidate genes in pediatric pilocytic astrocytoma. A, Graphical workflow: in step 1, OIS-controlling candidate genes were identified from published SASP factors (n = 62), from publicly available OIS datasets (n = 332), and related upregulated IPA pathway genes (n = 36). In step 2, the candidate gene lists were screened for genes upregulated in primary pilocytic astrocytoma (PA), as compared with normal brain. In step 3, a final OIS-controlling candidate gene list was generated by filtering for consensus in all three datasets and for druggability (n = 3). B, Boxplot of log₂ mRNA expression of the final OIS-controlling candidate genes in primary pilocytic astrocytomas (n = 182) versus normal brain (NB; n = 5), analyzed in R2. Depicted are median (black bar), quartiles (box), median ± 1.5 IQR (interquartile range; whiskers), and outliers (circles). C, Boxplot of log₂ mRNA expression of OIS-controlling candidate genes in the murine pilocytic astrocytoma model (n = 8) versus normal brain (NB) of healthy control mice (n = 8). Depicted are median (black bar), quartiles (box), median ± 1.5 IQR (whiskers), and outliers (circles). D, Boxplot of log2 mRNA expression of OIS-controlling candidate genes in DKFZ-BT66 cells in OIS (n = 3) versus proliferation (n = 3), as measured by gene expression profiling. Depicted are median (black bar), quartile (box), and median ± 1.5 IQR (whiskers). E, Fold change of IL1B and IL6 transcript levels measured by qRT-PCR during induction of OIS in DKFZ-BT66 cells in comparison with expression levels of DKFZ-BT66 cells in proliferation (= day 0). Depicted are mean ± SD of three independent experiments. F, IL1B and IL6 protein secretion measured in the supernatant of DKFZ-BT66 cells upon OIS induction by ELISA. Concentration was normalized to cell number on the day of collection (pg/cell). Depicted are mean ± SD of three independent experiments. Significant differences are indicated as *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student t test).

The first candidate gene list (“SASP genes”) consists of the SASP genes published by Coppé (n = 62 genes; ref. 17), accounting for the published SASP genes. The second candidate gene list (“Published OIS genes”) was generated by screening publicly available GEP datasets from six human OIS models (including RAS or BRAF^V600E–transduced fibroblasts and primary melanocytes) for genes upregulated in all 6 datasets (n = 332 genes). This list accounts for genes upregulated in independent general OIS models. The third gene list ("Top IPA pathway genes”) was generated by identifying the top signaling pathways upregulated in the “Published OIS genes” set performing IPA and including all genes related to the respective pathways (n = 36 genes). This list additionally adds all genes related to the pathways upregulated in OIS, which may not have been included on a single gene basis. In step 2 the expression of all three candidate gene lists (“SASP genes,” “Published OIS genes,” “Top IPA pathway genes”) was analyzed in n = 182 pilocytic astrocytoma samples (Supplementary Table S4) and compared with n = 5 normal brain samples, to identify the candidate genes relevant to pilocytic astrocytoma. In a third step, we filtered for consensus candidate genes (n = 5), by omitting all genes that did not appear in all three candidate gene lists. Finally, for experimental as well as therapeutic purposes, only genes which could be targeted by a small-molecule inhibitor or antibody were considered, resulting in a pilocytic astrocytoma–specific OIS-controlling candidate gene list of n = 3 genes (IL1B, IL6, TNFRSF1B; Fig. 2A and B). The candidates were validated in the murine in vivo pilocytic astrocytoma model (12) and found to be upregulated compared with healthy control animals (Fig. 2C). The pilocytic astrocytoma tumors induced by BRAF^V600E expression in neural progenitor cells showed upregulated expression of CDKN2A on mRNA level compared with healthy controls (Supplementary Fig. S1A), and cells derived from the pilocytic astrocytoma tumors were positive for SA-ß-Gal staining (Supplementary Fig. S1B). These data indicate that the murine pilocytic astrocytoma model indeed displays markers of senescence and SASP expression.

While the increased mRNA expression was validated by gene expression profiling in DKFZ-BT66 in OIS for all three OIS-controlling candidate genes (Fig. 2D), qRT-PCR during OIS induction could confirm the mRNA increase only for IL1B and IL6 but not for TNFRSF1B (Fig. 2E). IL1B and IL6 are both secreted into the supernatant, as detected by ELISA (Fig. 2F). Upregulation of IL1A/B and IL6 has previously been described in other senescence models in fibroblasts (18, 39–41), human epithelial cells (16), BRAF^V600E-transduced human melanocytes (18), and prostate epithelial cancer cells (16), suggesting that IL1B and IL6 are important regulators of OIS. We therefore focused on IL1B and IL6 as OIS-controlling candidate genes in pilocytic astrocytoma in the further analyses.

IL1 and IL6 signaling pathways are present and regulated during OIS in pilocytic astrocytoma

As we detected upregulation and secretion of IL1B and IL6 during induction of OIS, we next aimed at elucidation of their function during OIS in pilocytic astrocytoma. In a first step, the expression and activation of the IL1B and IL6 signaling cascades were determined in DKFZ-BT66 and in primary pediatric pilocytic astrocytoma tumors. The IL1R1 receptor is expressed on mRNA and protein level in DKFZ-BT66 cells (Fig. 3A and B), but no significant regulation was detected. While the activity of IL1B signaling is regulated by a variety of mechanisms, its receptor is indeed rarely upregulated in disease models (42). To determine activation of the IL1 pathway, protein expression of the downstream targets IRAK1 and phosphorylation of RELA was investigated. IRAK1, the interleukin-1 receptor-associated kinase 1, is phosphorylated and degraded upon activation of the IL1 pathway (43). Degradation of IRAK1 was indeed observed after induction of senescence and subsequent growth arrest in DFKZ-BT66 cells (Fig. 3B). NFκB, a transcription factor highly associated with OIS, has been shown to be an important regulator of the SASP (44, 45). Activation of NFκB was assessed by phosphorylation status of RELA, which increased after OIS induction in DKFZ-BT66 cells (Fig. 3B). In conclusion, the IL1 pathway is active during OIS in the DKFZ-BT66 cell model.

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Figure 3.

Functional validation of the IL1 and IL6 signaling pathways during OIS. A, Fold change of IL1R1 transcript levels were measured by qRT-PCR in DKFZ-BT66 upon OIS induction relative to levels during proliferation (=day 0). Depicted are mean ± SD of three independent experiments. B, IL1 pathway activity was determined by protein levels of IL1R1, IRAK1, phospho-RELA, and RELA in DKFZ-BT66 upon OIS induction measured by Western blot analysis. Actin serves as loading control. C, Fold change of IL6Ra transcript levels as measured by qRT-PCR in DKFZ-BT66 upon OIS induction relative to levels during proliferation (=day 0). Depicted are mean ± SD of three independent experiments. D, IL6 pathway activity was determined by protein levels of IL6Ra and pSTAT3/STAT3 upon OIS induction measured by Western blot analysis. Actin serves as loading control. E, Presence of IL1B and IL6 protein was detected by multiplex assay in all studied fresh frozen primary pilocytic astrocytoma (PA) samples (black; n = 22) and in normal fetal brain (gray; n = 1). Depicted is mean ± SD. Dots indicate values of individual samples.

The IL6Ra receptor is expressed on mRNA and protein level in DKFZ-BT66 cells (Fig. 3C and D). While no significant changes were observed on mRNA level (Fig. 3C), the protein levels decreased during OIS induction (Fig. 3D). It has been described that long-term activation of the IL6 pathway initiates a negative feedback loop, leading to internalization and degradation of IL6Ra (46). In parallel, we detected reduction of IL6 signaling during OIS induction in DKFZ-BT66 cells as measured by reduced phosphorylation of the downstream target STAT3 (p-STAT3) in comparison with initial levels (Fig. 3D). Activation of the IL6 pathway leads to phosphorylation of STAT3 within minutes (47), while long-term activation induces negative feedback and leads to downregulation of p-STAT3 (48). We conclude that the initial activity of the IL6 pathway is downregulated in our pilocytic astrocytoma cell culture model, possibly due to continuous IL6 secretion during induction of OIS and subsequent growth arrest.

Expression of IL1B and IL6 protein was analyzed in n = 22 pediatric pilocytic astrocytoma samples and one normal brain sample by multiplex assay and both cytokines were detected in every sample (Fig. 3E).

Taken together, these data indicate that the IL1 as well as the IL6 pathway is regulated during OIS in our pilocytic astrocytoma model, but only the IL1 pathways remains activated while a negative feedback regulation of the IL6 pathway is observed. Both cytokines are present in primary pediatric pilocytic astrocytoma.

IL1B, but not IL6 signaling, reduces proliferation of pilocytic astrocytoma cells and induces SASP expression

After observing elevated IL1B and IL6 levels during OIS, we next determined the role of both cytokines for OIS induction. Increasing concentrations of recombinant cytokines rIL1B and rIL6 were used to actively stimulate both pathways in proliferating DKFZ-BT66 cells.

Treatment with rIL1B resulted in significant reduction of proliferation of DKFZ-BT66 cells in a concentration-dependent manner, without affecting cell viability (Fig. 4A; Supplementary Fig. S2). Increasing concentrations of rIL1B above 100 pg/mL could not maximize the resulting growth reduction. The IL1 pathway was activated by rIL1B treatment, evident by degradation of IRAK1 as well as elevated protein levels of the precursor form of IL1B (31 kDa; Fig. 4B). Elevation of IL1B due to de novo translation after positive feedback activation has been described previously (49). Of note, the maximum reduction of both proliferation and of IRAK1 protein was seen at the same concentration of rIL1B (100 pg/mL), indicating a strong correlation between IL1 pathway activity and cell proliferation. On the contrary, no significant effect on proliferation of DKFZ-BT66 cells was detected upon treatment with rIL6 (Fig. 4C). The IL6 pathway was indeed activated after short-term treatment with rIL6, as observed by elevated phosphorylation of STAT3 in a concentration-dependent manner, excluding unsuccessful stimulation of the pathway (Fig. 4D).

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Figure 4.

IL1B signaling contributes to reduced pilocytic astrocytoma (PA) cell proliferation and induces expression of SASP factor. A, Cell count of proliferating DKFZ-BT66 cells under rIL1B treatment in concentrations indicated for 20 days. Depicted are mean ± SD of three independent experiments. Significant differences are indicated as *, P < 0.05 (Student t test). B, Protein levels of IRAK1 and pro-IL1B were determined by Western blot analysis under stimulation with rIL1B in the depicted concentrations for 4 hours in DKFZ-BT66 cells in proliferation. Actin serves as loading control. C, Cell count of proliferating DKFZ-BT66 cells under rIL6 treatment in concentrations indicated for 20 days. Depicted are mean ± SD of three independent experiments. D, Protein levels of pSTAT3 and STAT3 were determined by Western blot analysis under stimulation with rIL6 in the depicted concentrations for 15 minutes in DKFZ-BT66 cells in proliferation. Actin serves as loading control. E, Cell count under combination treatment with rIL1B and rIL6 in the depicted concentrations for 20 days. Depicted are mean ± SD of three independent experiments. Significant differences are indicated as *, P < 0.05 (Student t test). F, Protein levels of IRAK1, CDKN1A, and SV40 measured by Western blot analysis after long-term treatment with rIL1B (500 pg/mL) (+) for 0, 5, 10, and 20 days versus solvent control (−) treatment in DKFZ-BT66 in proliferation. Actin serves as loading control. G, Light microscopic comparison of DKFZ-BT66 cells grown under treatment with 500 pg/mL rIL1B versus solvent control at day 20. H, Barcode plot of GSEA depicting significant upregulation of the SASP genes in DKFZ-BT66 cells under treatment with 500 pg/mL rIL1B and doxycycline for five days versus DKFZ-BT66 cells in proliferation.

As the SASP is a complex mixture of inflammatory signaling molecules acting in a concerted fashion, we tested whether combination treatment of rIL1B and rIL6 has an additional or synergistic effect on cell proliferation. Cotreatment resulted in a significant downregulation of cell growth in comparison with solvent control, however, depending only on the concentration of rIL1B (Fig. 4E). Addition of rIL6 in increasing concentrations did not lead to additional reduction in cell growth.

The IL1 pathway was active for the entire duration (up to day 20) of the experiment as detected by IRAK1 degradation (Fig. 4F). SV40 large T antigen was expressed throughout the experiment and its downstream signaling resulted in reduced expression of CDKN1A (Fig. 4F). Thus, the inhibitory effect of IL1B on cell proliferation is not based on alterations in SV40 large T antigen and subsequent CDKN1A expression. As previously described, SA-β-Gal cannot be used as senescence marker in DKFZ-BT66 (14). However, enlarged cellular morphology characteristic for senescence was observed after treatment with rIL1B (Fig. 4G). In addition, significant upregulation of the SASP factors was determined by GEP under treatment with rIL1B (Fig. 4H).

In summary, IL1B, but not IL6, induces growth arrest of proliferating pilocytic astrocytoma cells, and IL1B induces senescent morphology and upregulation of the SASP factors. We conclude that IL1B plays an important role in induction of SASP-mediated OIS in pilocytic astrocytoma.

Interference with inflammatory signaling, but not with IL1 signaling alone, leads to suppression of the SASP and growth of senescent pilocytic astrocytoma cells

Having identified IL1B as important for senescence induction, we next assessed the role of IL1B for OIS maintenance. Senescent DKFZ-BT66 cells were treated with anakinra, an antagonist of the IL1 receptor. OIS-induced growth arrest could not be circumvented by treatment with anakinra alone, as determined by cell counts over the course of 20 days (Fig. 5A). IL1B signaling was inhibited, as shown by rescued IRAK1, pro-IL1B, and p-RELA levels (Fig. 5B). Pharmacologic inhibition of the IL1 signaling pathway alone therefore cannot bypass OIS in our model, similar to reports in other OIS models (18).

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Figure 5.

Inhibition of inflammatory signaling during OIS, but not IL1B alone, suppresses the SASP and leads to bypass of OIS. A, Cell count of senescent DKFZ-BT66 cells under anakinra treatment in the depicted concentrations for 20 days. Cells were cultured without doxycycline 5 days prior to treatment as well as throughout the duration of the entire experiment. Shown are mean ± SD of three independent experiments. B, Protein levels of IRAK1, pro-IL1B, p-RELA, and RELA determined by Western blot analysis in DKFZ-BT66 cells in OIS treated with rIL1B (500 pg/mL) ± anakinra (20 μg/mL) for 4 hours. Actin serves as loading control. C, Cell count of senescent DKFZ-BT66 cells treated with 100 nmol/L dexamethasone (Dexa) or solvent control (0 nmol/L Dexa) for 20 days. Cells were cultured without doxycycline 5 days prior to treatment as well as throughout the duration of the entire experiment. Depicted are mean ± SD of three independent experiments. Significant differences are indicated as *, P < 0.05; ***, P < 0.001 (Student t test). D, Protein levels of IRAK1 determined by Western blot analysis in DKFZ-BT66 cells in OIS treated with 100 nmol/L dexamethasone (Dexa) (+) or solvent control (−) for 0, 5, 10, and 20 days. Actin serves as loading control. E, Barcode plot of GSEA reveals significant downregulation of the SASP genes in DKFZ-BT66 cells in OIS under treatment with dexamethasone (Dexa) for five days. F, IPA analysis of GEPs of DKFZ-BT66 cells in OIS treated with 100 nmol/L dexamethasone (n = 3 replicates) compared with proliferating DKFZ-BT66 cells (n = 3 replicates; red, OIS + Dexa), or DKFZ-BT66 cells in proliferation treated with 500 pg/mL rIL1B (n = 3 replicates) compared with DKFZ-BT66 cells in OIS (n = 3 replicates; blue, proliferation + rIL1B) for five days. The 4 common genes of the top 5 upstream regulators of each condition are displayed. The z-score predicts the activation status of the upstream regulator, positivity indicates activation, negativity indicates inhibition. Color indicates from which condition the z-score was calculated. White bar: z-score for TREM1 in the OIS + Dexa condition did not pass the threshold to indicate its inhibition/activation.

As inhibition of single SASP factors did not suffice to overcome growth arrest of pilocytic astrocytoma cells in OIS, we attempted to bypass OIS by treatment with dexamethasone, a broad anti-inflammatory drug, known to inhibit the SASP (50, 51). Treatment of senescent DKFZ-BT66 cells with 100 nmol/L dexamethasone for 20 days resulted in a significant increase in cell proliferation compared with solvent control (Fig. 5C). Exemplary for SASP factors, dexamethasone strongly inhibited the IL1 pathway as determined by rescued protein levels of IRAK1 (Fig. 5D). Treatment of DKFZ-BT66 cells in OIS with dexamethasone for five days led to a significant reduction in expression of SASP factors as determined by GEP, illustrated in the GSEA (Fig. 5E). IPA analysis of the GEPs revealed IL1B as the top upstream regulator, predicted to be inhibited under dexamethasone treatment (Supplementary Table S5). Importantly, of the top five upstream regulators identified by IPA in either senescent DKFZ-BT66 cells treated with dexamethasone or in proliferating DKFZ-BT66 cells treated with rIL1B, 4 of 5 were identical (IL1B, TREM1, TNF, NFκB complex) and regulated in an opposite fashion (inhibition vs. activation; Fig. 5F). This suggests similar SASP pathways to be involved in IL1B-induced senescence and reversal of senescence by dexamethasone, in an opposite manner. In summary, these data indicate that bypass of SASP-mediated OIS-induced growth arrest is possible by inhibiting multiple SASP pathways with an anti-inflammatory drug in pilocytic astrocytoma cells.

IL1B and the SASP are expressed in primary pilocytic astrocytomas and predict favorable progression-free survival independent of tumor resection status

The SASP as well as IL1B play a significant role in the regulation of OIS in our pilocytic astrocytoma cell model and could mediate the variable growth behavior of pediatric pilocytic astrocytomas observed clinically. To assess the clinical relevance of our findings we analyzed the expression of IL1B and the SASP in primary pediatric pilocytic astrocytoma samples. Using a multiplex assay, all n = 14 SASP factors present in the assay were detected in primary pilocytic astrocytomas on protein level (Supplementary Fig. S3). To test for a correlation between SASP factor mRNA expression and clinical outcome in pilocytic astrocytoma patients, the ICGC PedBrain pilocytic astrocytoma cohort [n = 112 patients; survival data available for 110/112 patients (98.2%; ref. 3), for annotations see Supplementary Table S4] was analyzed for expression of IL1B as well as by means of a SASP sum score (SASP score), which sums up the expression levels of all SASP factors for one patient. Increased expression of IL1B alone (both as continuous or grouped variable according to median cutoff) predicted favorable prognosis (e.g., continuous variable: HR = 0.4, 95% Cl, 0.24–0.69, P = 0.0008; n = 110 patients, Supplementary Table S6). IL1B remained a factor for good prognosis in a multivariate analysis after accounting for other significant prognostic factors such as extent of tumor resection (HR = 0.37, 95% Cl, 0.18–0.75, P = 0.0056; n = 90 patients, Supplementary Table S6) or radiotherapy (HR = 0.35, 95% Cl, 0.15–0.77, P = 0.0079; n = 75 patients, Supplementary Table S6). Five-year progression-free survival (PFS) was 85% in the "IL1B high" group versus 46% in the "IL1B low" group, and survival differed significantly (log-rank test; Fig. 6A). Similarly, patients with a higher SASP score (continuous variable) had a significantly more favorable PFS (HR = 0.56, 95% CI, 0.34–0.93, P = 0.026, n = 110 patients; Supplementary Table S6). The prognostic effect of the SASP score remained significant in a multivariate analysis after accounting for other significant prognostic factors such as extent of resection (HR = 0.36, 95% CI, 0.16–0.82, P = 0.01; n = 90, Supplementary Table S6) or radiotherapy (HR = 0.19, 95% CI, 0.06–0.52, P = 0.0006; n = 75 patients, Supplementary Table S6), or both (HR = 0.19, 95% CI, 0.03–0.72, P = 0.01; n = 58 patients, Supplementary Table S6). When patients were grouped into three groups according to SASP score tertiles, the 5-year PFS was 48% for the "SASP low" group, 61% for the "SASP intermediate" group, and 90% for the "SASP high" group, and survival differed significantly (log-rank test; Fig. 6B). Because resection status is a strong prognostic factor, we analyzed PFS separately for either gross total resection (GTR) or subtotal resection (STR). The 5-year PFS was 100% for patients with GTR in the "SASP high" and “SASP intermediate” group versus 63.3% in the "SASP low" group, and survival differed significantly (log-rank test; Fig. 6C). On the contrary, patients with STR in the "SASP low" group showed a particularly poor 2- and 3-year PFS of 52% and 0%, respectively (Fig. 6D).

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Figure 6.

SASP factor expression predicts PFS independent of resection status implying a crucial role of inflammatory signaling for pilocytic astrocytoma (PA) tumor growth behavior. A, Kaplan–Meier analysis of pilocytic astrocytoma patients (n = 110) depicting superior progression-free survival (PFS) in the IL1B mRNA high expression group ("IL1B high"; P = 0.006, log-rank). B, Kaplan–Meier analysis with pilocytic astrocytoma patients (n = 110) grouped into three groups according to SASP score tertiles, depicting significantly different PFS (P = 0.02, log-rank). C, Kaplan–Meier analysis, only patients with pilocytic astrocytoma with gross total resection (GTR; n = 61) are shown depicting significantly different PFS (P = 0.047, log-rank). Both “SASP high” and “SASP intermediate” have no events and curves are fully overlapping. D, Kaplan–Meier analysis, only patients with subtotal resection (STR; n = 32) are shown. Panel A–D all depict patients from the same pilocytic astrocytoma cohort from ICGC PedBrain (PFS available for n = 110/112, 98.2%). Information on resection status was available for n = 93/110 (84.5%) patients of the ICGC pilocytic astrocytoma cohort.

In conclusion, SASP factors are expressed on mRNA and protein level in primary pediatric pilocytic astrocytomas. Elevated mRNA expression of SASP factors correlates with a high probability to remain progression-free, and low expression of SASP factors correlates with a high risk of recurrence, especially in subtotally resected cases.

Senescent DKFZ-BT66 cells are more responsive to senolytic BCL2 family inhibitors than proliferating cells

The low proliferation index observed in pilocytic astrocytomas (15) suggests that most of the tumor cells persist in a senescent state and thus are unlikely to respond to standard chemotherapeutic agents relying on cell division for their effect. To investigate the targetability of senescence in pilocytic astrocytoma, we performed a drug screen in DKFZ-BT66 cells in OIS and proliferation testing single agents and combination treatments of several senolytic agents, standard-of-care chemotherapeutics, and a MEK inhibitor. Senescent DKFZ-BT66 cells showed increased sensitivity to navitoclax and ABT-737 (both BCL2 family inhibitors) in comparison with proliferating cells. We did not observe elevated sensitivity to the combination of dasatinib and quercetin (Fig. 7A–C), as has been previously described for other senescent cells (52). Primary human astrocytes, which served as control, responded at high concentrations only (Supplementary Fig. S4A–S4G). No significant response to carboplatin, vincristine, or trametinib was detected in senescent DKFZ-BT66 cells (Fig. 7D–F). In contrast to carboplatin, vincristine reduced metabolic activity in proliferating DKFZ-BT66 cells. As the SV40 large T antigen inhibits TP53 signaling in proliferating DKFZ-BT66 cells, carboplatin cannot induce TP53-dependent apoptosis. Therefore, the lack of effect is expected and due to the specifics of this model, but not indicative of resistance. An increase in metabolic activity in both proliferating and senescent DKFZ-BT66 cells was observed under trametinib treatment, as we have described previously (14).

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Figure 7.

Senescent DKFZ-BT66 cells respond to senolytic agents. A–F, Assessment of metabolic activity by CellTiter-Glo of senescent (red) or proliferating (blue) DKFZ-BT66 cells treated for 72 hours with navitoclax (A), ABT-737 (B), dasatinib plus quercetin (C), carboplatin (D), vincristine (E), and trametinib (F) in the indicated concentrations. Shown are mean ± SD of three technical replicates. IC₅₀ concentrations are depicted for DKFZ-BT66 cells in OIS (red) and in proliferation (blue). NA, not available. G–I, Assessment of metabolic activity by CellTiter-Glo of senescent (red) or proliferating (blue) DKFZ-BT66 cells treated for 72 hours with navitoclax in combination with carboplatin (G), vincristine (H), or trametinib (I) in the indicated concentrations. Shown are mean ± SD of three technical replicates.

Navitoclax is currently being tested in several clinical trials and was evaluated as safe and well tolerated with dose-dependent thrombocytopenia as major adverse event (53). We therefore investigated the combination of navitoclax with standard-of-care treatment for patients with pilocytic astrocytoma. In our screen, we observed additive effects in the combination treatments compared with carboplatin, vincristine, or trametinib alone. No antagonistic effect was detected (Fig. 7G–I). Similar results were observed for ABT-737 in combination treatments (Supplementary Fig. S4H–S4J). In conclusion, addition of a BCL2 family inhibitor to the standard-of-care treatment regimen for patients with pilocytic astrocytoma could help eradicate senescent pilocytic astrocytoma cells, which do not respond to chemotherapy and might be the source of tumor recurrence or progression at a later time point.