Supplementary Figure 2 from Genome-Wide Allelic Imbalance Analysis of Pediatric Gliomas by Single Nucleotide Polymorphic Allele Array

Repurposing approved non-antitumor drugs is a promising and affordable strategy in drug discovery to identify new therapeutic uses different from the original medical indication that may help increase the number of possible, effective anticancer drugs. The use of drugs in ways other than their original FDA-approved indications could offer novel avenues such as bypassing the chemoresistance and recurrence seen with conventional therapy and treatment; moreover, it can offer a safe and economic strategy for combination therapy. Recent works have demonstrated the anticancer properties of the FDA-approved drug Mebendazole. This synthetic benzimidazole proved effective against a broad spectrum of intestinal Helminthiasis. Mebendazole can penetrate the blood–brain barrier and has been shown to inhibit the malignant progression of glioma by targeting signaling pathways related to cell proliferation, apoptosis, or invasion/migration, or by increasing the sensitivity of glioma cells to conventional chemotherapy or radiotherapy. Moreover, several preclinical models and ongoing clinical trials explore the efficacy of Mebendazole in multiple cancers, including acute myeloid leukemia, brain cancer, oropharyngeal squamous cell carcinoma, breast cancer, gastrointestinal cancer, lung carcinoma, adrenocortical carcinoma, prostate cancer, and head and neck cancer. The present review summarizes central literature regarding the anticancer effects of MBZ in cancer cell lines, animal tumor models, and clinical trials to suggest possible strategies for safe and economical combinations of anticancer therapies in brain cancer. Mebendazole might be an excellent candidate for the treatment of brain tumors because of its efficacy both when used as monotherapy and in combination as an enhancement to standard chemotherapeutics and radiotherapy, due to its effectiveness on tumor angiogenesis inhibition, cell cycle arrest, apoptosis induction, and targeting of critical pathways involved in cancer such as Hedgehog signaling. Therefore, attention to MBZ repurposing has recently increased because of its potential therapeutic versatility and significant clinical implications, such as reducing medical care costs and optimizing existing therapies. Using new treatments is essential, particularly when current therapeutics for patients with brain cancer fail.

Supplementary Tables 1-2 from Genome-Wide Allelic Imbalance Analysis of Pediatric Gliomas by Single Nucleotide Polymorphic Allele Array

Characterization of ependymoma lines (S1); Nested two-round PCR for detection of SEC61G-EGFR gene fusion (S2); Expression of mutant EGFR in patient- and xenograft-derived lines (S3); EGFR signaling in MD and MI ependymoma lines and effects of EGFR TKIs (S4); antiproliferative effects of EGFR TKIs in EPV-FL and EPV-FL-MI lines (S5); EGFR labeling of intracranial EPP and EPP-MI tumor sections of AEE788-administered mice (S6).

Supplementary Figure 1 from Genome-Wide Allelic Imbalance Analysis of Pediatric Gliomas by Single Nucleotide Polymorphic Allele Array

Fusion transcripts identified by ChimeraScan in EPP-MI line

IC50 values of EGFR TKIs gefitinib and AEE788 in paired MD and MI ependymoma lines

Childhood ependymomas are heterogenous chemoresistant neoplasms arising from aberrant stem-like cells. Epigenome deregulation plays a pivotal role in ependymoma pathogenesis, suggesting that epigenetic modifiers hold therapeutic promise against this disease. Bromodomain and extraterminal domain (BET) proteins are epigenome readers of acetylated signals in histones and coactivators for oncogenic and stemness-related transcriptional networks, including MYC/MYCN (Proto-Oncogene, BHLH Transcritpion Factor)-regulated genes. We explored BET inhibition as an anticancer strategy in a panel of pediatric patient-derived ependymoma stem cell models by OTX015-mediated suppression of BET/acetylated histone binding. We found that ependymoma tissues and lines express BET proteins and their targets MYC and MYCN. In vitro, OTX015 reduced cell proliferation by inducing G0/G1-phase accumulation and apoptosis at clinically tolerable doses. Mechanistically, inhibitory p21 and p27 increased in a p53-independent manner, whereas the proliferative driver, phospho-signal transducer and activator of transcription 3 (STAT3), decreased. Upregulation of apoptosis-related proteins and survivin downregulation were correlated with cell line drug sensitivity. Minor alterations of MYC/MYCN expression were reported. In vivo, OTX015 significantly improved survival in 2/3 orthotopic ependymoma models. BET proteins represent promising targets for pharmaceutical intervention with OTX015 against ependymoma. The identification of predictive determinants of sensitivity may help identify ependymoma molecular subsets more likely to benefit from BET inhibitor therapies.

The neurotrophin nerve growth factor (NGF) modulates the growth of human gliomas and is able to induce cell differentiation through the engagement of tropomyosin receptor kinase A (TrkA) receptor, although the role played in controlling glioma survival has proved controversial. Unfortunately, the slow growth rate of low-grade gliomas (LGG) has made it difficult to investigate NGF effects on these tumors in preclinical models. In fact, patient-derived low-grade human astrocytoma cells duplicate only a limited number of times in culture before undergoing senescence. Nevertheless, replicative senescence can be counteracted by overexpression of hTERT, the catalytic subunit of telomerase, which potentially increases the proliferative potential of human cells without inducing cancer-associated changes. We have extended, by hTERT transduction, the proliferative in vitro potential of a human LGG cell line derived from a pediatric pilocytic astrocytoma (PA) surgical sample. Remarkably, the hTERT-transduced LGG cells showed a behavior similar to that of the parental line in terms of biological responses to NGF treatment, including molecular events associated with induction of NGF-related differentiation. Therefore, transduction of LGG cells with hTERT can provide a valid approach to increase the in vitro life-span of patient-derived astrocytoma primary cultures, characterized by a finite proliferative potential.

The Ras/Raf/MEK/MAPK signaling cascade is frequently activated in human cancer and serves a crucial role in the oncogenesis of pediatric low‑grade gliomas (PLGGs). Therefore, drugs targeting kinases among the mitogen‑activated protein kinase (MAPK) effectors of receptor tyrosine kinase signaling may represent promising candidates for the treatment of PLGGs. The aim of the present study was to elucidate the anticancer effects of the MEK inhibitor Selumetinib on two low‑grade glioma cell lines and the possible underlying effects on intracellular signal transduction. The two cancer cell lines displayed different levels of sensitivity to Selumetinib, as Res186 cells were resistant (IC50>1 µM), whereas Res259 cells were sensitive (IC50≤1 µM) to MEK inhibition. Despite the different levels of sensitivity, Selumetinib mediated the phosphorylation of AKT and MEK in both cell lines and suppressed the phosphorylated MAPK cascades. In addition, Selumetinib induced cell cycle arrest at the G0/G1 phase by downregulating the expression levels of cyclin D1 and p21 and upregulating those of p27 compared with those in the control cells. A Res259 cell line with acquired resistance to Selumetinib (Res259/R) was next established and biologically and molecularly characterized, and it was demonstrated that addition of a selective cAMP‑dependent protein kinase A inhibitor to Selumetinib overcame drug resistance in Res 259/R cells. In conclusion, the results of the present study provided three low‑grade glioma cell line models characterized by sensitivity, intrinsic and acquired resistance to Selumetinib, which may be usuful tools to study new mechanisms of chemoresistance to MEK inhibitors and to explore alternative therapeutic strategies in low‑grade gliomas for personalization of treatment.

Objective The neurotrophin nerve growth factor (NGF) affects survival, regulation and differentiation of both central and peripheral nervous system neurons. NGF exerts its effects primarily through tropomyosin receptor kinase A (TrkA), inducing a cascade of tyrosine kinase-initiated responses. In spite of its importance, the general behavior of NGF looks contradictory: its effects can be both stimulatory and inhibitory. The present study aims to explore the molecular mechanisms induced by NGF in glioma cancer cells. Methods The effects of NGF were investigated in high grade glioma and low grade pediatric glioma (PLGG) cell lines through comparative studies. In particular, we investigated TrkA-mediated cellular pathways, molecular signaling, proliferation, cell cycle and cellular senescence. Results We found that exposure of PLGG cells to NGF produced stable growth arrest with the features of a senescence phenotype but without the expression of anti-poly(ADP-ribose) polymerase cleavage, a marker of apoptosis. Moreover, NGF treatment promoted the phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), signal transducer and activator of transcription 3 (STAT3), and phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling. In addition, K252a, a TrkA inhibitor, significantly reduced the phosphorylation of the aforementioned signaling pathways, suggesting that NGF-activated ERK1/2 and AKT signaling take place downstream of TrkA-neurotrophin interaction. Conclusions These findings provide the first evidence that NGF can induce senescence of PLGG cells in a receptor-mediated fashion, thus supporting the hypothesis that in the clinical setting NGF might be beneficial to pediatric glioma patients.

Abstract The basis for molecular and cellular heterogeneity in ependymomas of the central nervous system is not understood. This study suggests a basis for this phenomenon in the selection for mitogen-independent (MI) stem-like cells with impaired proliferation but increased intracranial tumorigenicity. MI ependymoma cell lines created by selection for EGF/FGF2-independent proliferation exhibited constitutive activation of EGFR, AKT, and STAT3 and sensitization to the antiproliferative effects of EGFR tyrosine kinase inhibitors (TKI). One highly tumorigenic MI line harbored membrane-bound, constitutively active, truncated EGFR. Two EGFR mutants (ΔN566 and ΔN599) were identified as products of intrachromosomal rearrangements fusing the 3′ coding portion of the EGFR gene to the 5′-UTR of the SEC61G, yielding products lacking the entire extracellular ligand-binding domain of the receptor while retaining the transmembrane and tyrosine kinase domains. EGFR TKI efficiently targeted ΔN566/ΔN599-mutant–mediated signaling and prolonged the survival of mice bearing intracranial xenografts of MI cells harboring these mutations. RT-PCR sequencing of 16 childhood ependymoma samples identified SEC61G–EGFR chimeric mRNAs in one infratentorial ependymoma WHO III, arguing that this fusion occurs in a small proportion of these tumors. Our findings demonstrate how in vitro culture selections applied to genetically heterogeneous tumors can help identify focal mutations that are potentially pharmaceutically actionable in rare cancers. Cancer Res; 77(21); 5860–72. ©2017 AACR.

The male-to-female sex ratio for medulloblastoma (MB) is approximately 1.5∶1, female gender being also a favorable prognostic factor. This study aimed at evaluating the impact of gender on MB tumorigenesis.In vitro activity of 17β-estradiol (E2), DPN [2,3-bis(4-hydroxyphenyl)-propionitrile, a selective estrogen receptor β (ERβ)-agonist], PPT [4,4',4″-(4-Propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol, a selective ERα-agonist] or DHT (5 alpha-dihydrotestosterone) was evaluated in three human MB cell lines. D283 Med cells were transplanted into athymic mice.A significant expression of ERβ, with little or no ERα, and low AR (androgen receptor) was found in MB cell lines. The compounds tested did not affect cell proliferation. In vivo, we observed a significantly lower growth of D283 Med in nude female mice compared to males. At microscopic examination, tumors from females showed a shift towards differentiation, as evaluated by lower nestin, and higher NSE (neuron-specific enolase) and GFAP (glial fibrillary acidic protein) expression compared to males. Tumors from females also showed lower Ki67 and p53 expression. The wild-type ERβ protein (ERβ1) was lost in male tumors, while it was a permanent feature in females, and a strong negative correlation was found between Ki67 and ERβ1 expression. Conversely, tumor levels of ERβ2 and ERβ5 did not significantly differ between genders. Increased levels of cyclin-dependent kinase inhibitor p21 were observed in females, suggesting that estrogen may decrease tumor growth through blocking cell cycle progression. An inhibition of the insulin-like growth factor I (IGF-I) signaling was also evident in females.We provides mechanistic evidence supporting the idea that ERβ1 signaling may have pro-differentiation and tumor suppressive function in medulloblastomas.

BACKGROUNDEpendymoma management remains challenging because of the inherent chemoresistance of this tumor. To determine whether ependymoma stem cells (SCs) might contribute to therapy resistance, we investigated the sensitivity of ependymoma SCs to temozolomide and etoposide.METHODSThe efficacies of the two DNA damaging agents were explored in two ependymoma SC lines in vitro and in vivo models.RESULTSEpendymoma SC lines were highly sensitive to temozolomide and etoposide in vitro, but only temozolomide impaired tumor-initiation properties. Consistently, temozolomide but not etoposide showed significant antitumoral activity on ependymoma SC-driven subcutaneous and orthotopic xenografts by reducing the mitotic fraction. In vitro temozolomide at the EC50 (10 µM) induced accumulation of cells in the G2/M phase that was unexpectedly accompanied by downregulation of p27 and p21 without modulation of full-length p53 (FLp53). Differentiation-committed ependymoma SCs acquired resistance to temozolomide. Inhibition of proliferation was partly due to apoptosis, that occurred earlier in differentiated cells as compared to neurospheres. The activation of apoptosis correlated with an increase in p53β/γ isoforms without modulation of FLp53 under both serum-free and differentiation-promoting media. Incubation of cells in both conditions with temozolomide resulted in increased glioneuronal differentiation exhibiting elevated glial fibrillary acidic protein, galactosylceramidase, and βIII-tubulin expression compared to untreated controls. O(6)-methylguanine DNA methyltransferase (MGMT) transcript levels were very low in SCs, and were increased by treatment and, epigenetically, by differentiation through MGMT promoter unmethylation.CONCLUSIONEpendymoma growth might be impaired by temozolomide through preferential depletion of a less differentiated, more tumorigenic, MGMT-negative cell population with stem-like properties.

Some lines of evidence suggest that tumors, including ependymoma, might arise from a subpopulation of cells, termed cancer stem cells (CSCs), with self-renewal and tumor-initiation properties. Given the strict dependence of CSCs on epidermal growth factor (EGF) through EGF receptor (EGFR), we investigated the effects of EGFR inhibitors in ependymoma-stem cells (SCs) in vitro and in orthotopic mouse models. We established two ependymoma-SC lines from two recurrent pediatric ependymoma. Both lines expressed markers of radial glia--the candidate SCs of ependymoma--and showed renewal ability, multipotency, and tumorigenicity after orthotopic implantation, despite markedly different expression of CD133 (94 vs. 6%). High phosphorylated-EGFR/EGFR ratio was detected, which decreased after differentiation. EGFR inhibitors (gefitinib and AEE788) reduced clonogenicity, proliferation and survival of ependymoma-SC lines dose-dependently, and blocked EGF-induced activation of EGFR, Akt and extracellular signal-regulated kinase 1/2. Overall, AEE788 was more effective than gefitinib. EGFR blockade as well as differentiation strongly reduced CD133 expression. However, ex vivo treatment with AEE788 did not impair orthotopic tumor engraftment, whereas ex vivo differentiation did, suggesting that CD133 does not absolutely segregate for tumorigenicity in ependymoma-SCs. Orally administered AEE788 prolonged survival of mice bearing ependymoma-SC-driven orthotopic xenografts from 56 to 63 days, close to statistical significance (log-rank p=0.06). Our study describes for the first time EGFR signaling in ependymoma-SCs and the effects of EGFR blockade in complementary in vitro and in vivo systems. The experimental models we developed can be used to further investigate the activity of EGFR inhibitors or other antineoplastic agents in this tumor.

The present study aimed to evaluate the new water soluble camptothecin analogue Namitecan (ST1968) in preclinical paediatric tumour models of the nervous system comprehensive of neuroblastoma, primitive neuroectodermal tumours/PNET and medulloblastoma where the drug was compared to Irinotecan.

Retinoic acid therapy is nowadays an important component of treatment for residual disease of stage IV neuroblastoma after multimodal therapy. Nevertheless, arising resistance and treatment toxicity could represent relevant limiting factors. In the present study, we show that retinoic acid enhances the cytostatic and apoptogenic properties of the novel adamantyl retinoid ST1926 in a panel of neuroblastoma cells with different p53 status and caspase 8 expression, resulting in synergistic effects as assessed by Combination Index and Isobologram analysis. Under conditions where the two drugs alone produced no toxic effects, their combination resulted in enhanced G2-M arrest and sub-G1 population as shown by BrdU pulse-chase and labeling experiments. PARP cleavage, caspase 3, 8 and 9 activation and modulation of DR4 and FAS were indicative of enhanced apoptosis triggered by the co-incubation of the two drugs whereas neither ST1926-mediated genotoxic damage nor ATRA-differentiating effects were affected by the combined treatment. Caspase-3 and 8-mediated apoptosis appeared to play an important role in the drugs synergism. In fact, the addition of a pan-caspase inhibitor ZVAD-FMK reverted this effect in SK-N-DZ cells, and synergism was confined to limited drugs doses in HTLA cells not expressing caspase-8. Although not modulated, p53 appeared to enhance cells responsiveness to retinoid/ATRA combination. In vivo studies in the most sensitive neuroblastoma model SK-N-DZ, confirmed enhanced activity of the drugs combination vs single treatments. The study provides important lines of evidence that such a drugs combination could represent a less toxic and more effective approach for maintenance treatment in children with neuroblastoma.

Namitecan (ST1968), a novel hydrophilic camptothecin analog of the 7-oxyiminomethyl series, was selected for clinical development on the basis of its promising preclinical efficacy. Since there is clinical evidence of efficacy of camptothecins against pediatric tumors, this study was performed to explore the antitumor and antiangiogenic activity of the camptothecin derivative in pediatric sarcoma models. With the exception of an undifferentiated rhabdomyosarcoma (A204), namitecan exhibited curative efficacy even at well-tolerated suboptimal doses in a panel of five models. The good therapeutic index of namitecan likely reflected a high and persistent drug accumulation at tumor site. The four responsive tumors were characterized by high topoisomerase I expression. In the RD/TE-671 rhabdomyosarcoma model the drug activity was associated with a marked antiangiogenic effect, which was consistent with the downregulation of proangiogenic factors, including VEGF, bFGF and the multifunctional chemokines CCL-2 and CXCL16. In agreement with this modulation, the combination of low doses of namitecan with other antiangiogenic agents, such as bevacizumab (a humanized anti-VEGF antibody) and sunitinib (a multitarget tyrosine kinase inhibitor effective against receptors implicated in the angiogenesis process), enhanced the antitumor effects. In conclusion, this preclinical study provides evidence of curative efficacy of namitecan at well-tolerated doses against pediatric sarcoma models, likely reflecting a contribution of antiangiogenic effects. Based on the promising therapeutic profile, namitecan is a good candidate for clinical evaluation in pediatric sarcomas.