Sustained cancer‐relevant alternative RNA splicing events driven by PRMT5 in high‐risk neuroblastoma

Protein arginine methyltransferase 5 (PRMT5) is over‐expressed in a wide variety of cancers and is implicated as having a key oncogenic role, achieved in part through its control of the master transcription regulator E2F1. We investigated the relevance of PRMT5 and E2F1 in neuroblastoma (NB) and found that elevated expression of PRMT5 and E2F1 occurs in poor prognosis high‐risk disease and correlates with an amplified Myelocytomatosis viral‐related oncogene, neuroblastoma‐derived (MYCN) gene. Our results show that MYCN drives the expression of splicing factor genes that, together with PRMT5 and E2F1, lead to a deregulated alternative RNA splicing programme that impedes apoptosis. Pharmacological inhibition of PRMT5 or inactivation of E2F1 restores normal splicing and renders NB cells sensitive to apoptosis. Our findings suggest that a sustained cancer‐relevant alternative RNA splicing programme desensitises NB cells to apoptosis, and identify PRMT5 as a potential therapeutic target for high‐risk disease

The HBP1 tumor suppressor is a negative epigenetic regulator of MYCN driven neuroblastoma through interaction with the PRC2 complex

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DNA-Dependent Protein Kinase As Molecular Target for Radiosensitization of Neuroblastoma Cells.

Tumor cells might resist therapy with ionizing radiation (IR) by non-homologous end-joining (NHEJ) of IR-induced double-strand breaks. One of the key players in NHEJ is DNA-dependent protein kinase (DNA-PK). The catalytic subunit of DNA-PK, i.e. DNA-PKcs, can be inhibited with the small-molecule inhibitor NU7026. In the current study, the in vitro potential of NU7026 to radiosensitize neuroblastoma cells was investigated. DNA-PKcs is encoded by the PRKDC (protein kinase, DNA-activated, catalytic polypeptide) gene. We showed that PRKDC levels were enhanced in neuroblastoma patients and correlated with a more advanced tumor stage and poor prognosis, making DNA-PKcs an interesting target for radiosensitization of neuroblastoma tumors. Optimal dose finding for combination treatment with NU7026 and IR was performed using NGP cells. One hour pre-treatment with 10 μM NU7026 synergistically sensitized NGP cells to 0.63 Gy IR. Radiosensitizing effects of NU7026 increased in time, with maximum effects observed from 96 h after IR-exposure on. Combined treatment of NGP cells with 10 μM NU7026 and 0.63 Gy IR resulted in apoptosis, while no apoptotic response was observed for either of the therapies alone. Inhibition of IR-induced DNA-PK activation by NU7026 confirmed the capability of NGP cells to, at least partially, resist IR by NHEJ. NU7026 also synergistically radiosensitized other neuroblastoma cell lines, while no synergistic effect was observed for low DNA-PKcs-expressing non-cancerous fibroblasts. Results obtained for NU7026 were confirmed by PRKDC knockdown in NGP cells. Taken together, the current study shows that DNA-PKcs is a promising target for neuroblastoma radiosensitization

Enhancer of zeste homologue 2 plays an important role in neuroblastoma cell survival independent of its histone methyltransferase activity

Neuroblastoma is predominantly characterised by chromosomal rearrangements. Next to V-Myc Avian Myelocytomatosis Viral Oncogene Neuroblastoma Derived Homolog (MYCN) amplification, chromosome 7 and 17q gains are frequently observed. We identified a neuroblastoma patient with a regional 7q36 gain, encompassing the enhancer of zeste homologue 2 (EZH2) gene. EZH2 is the histone methyltransferase of lysine 27 of histone H3 (H3K27me3) that forms the catalytic subunit of the polycomb repressive complex 2. H3K27me3 is commonly associated with the silencing of genes involved in cellular processes such as cell cycle regulation, cellular differentiation and cancer. High EZH2 expression correlated with poor prognosis and overall survival independent of MYCN amplification status. Unexpectedly, treatment of 3 EZH2-high expressing neuroblastoma cell lines (IMR32, CHP134 and NMB), with EZH2-specific inhibitors (GSK126 and EPZ6438) resulted in only a slight G1 arrest, despite maximum histone methyltransferase activity inhibition. Furthermore, colony formation in cell lines treated with the inhibitors was reduced only at concentrations much higher than necessary for complete inhibition of EZH2 histone methyltransferase activity. Knockdown of the complete protein with three independent shRNAs resulted in a strong apoptotic response and decreased cyclin D1 levels. This apoptotic response could be rescued by overexpressing EZH2ΔSET, a truncated form of wild-type EZH2 lacking the SET transactivation domain necessary for histone methyltransferase activity. Our findings suggest that high EZH2 expression, at least in neuroblastoma, has a survival function independent of its methyltransferase activity. This important finding highlights the need for studies on EZH2 beyond its methyltransferase function and the requirement for compounds that will target EZH2 as a complete protei

Mesoporous silica nanoparticles as drug delivery systems for targeted inhibition of notch signaling in cancer

Notch signaling, a key regulator of stem cells, is frequently overactivated in cancer. It is often linked to aggressive forms of cancer, evading standard treatment highlighting Notch as an exciting therapeutic target. Notch is in principle "druggable" by ¿-secretase inhibitors (GSIs), inhibitory peptides and antibodies, but clinical use of Notch inhibitors is restricted by severe side effects and there is a demand for alternative cancer-targeted therapy. Here, we present a novel approach, using imagable mesoporous silica nanoparticles (MSNPs) as vehicles for targeted delivery of GSIs to block Notch signaling. Drug-loaded particles conjugated to targeting ligands induced cell-specific inhibition of Notch activity in vitro and exhibited enhanced tumor retainment with significantly improved Notch inhibition and therapeutic outcome in vivo. Oral administration of GSI-MSNPs controlled Notch activity in intestinal stem cells further supporting the in vivo applicability of MSNPs for GSI delivery. MSNPs showed tumor accumulation and targeting after systemic administration. MSNPs were biocompatible, and particles not retained within the tumors, were degraded and eliminated mainly by renal excretion. The data highlights MSNPs as an attractive platform for targeted drug delivery of anticancer drugs with otherwise restricted clinical application, and as interesting constituents in the quest for more refined Notch therapies. © The American Society of Gene & Cell Therapy

NU7026 does not radiosensitize low DNA-PKcs-expressing non-cancerous cells.

<p>(A) Radiosensitizing effect of NU7026 in neuroblastoma cell lines NGP, SHSY5Y, SHEP2, SJNB12, LAN5 and SKNBE(2) compared with radiosensitizing effects in non-cancerous fast-proliferating fibroblast cell lines F1366, F1641, F0577 and F2112. Co-treated cells were exposed to 0.63 Gy IR after 1 h pre-treatment with 10 μM NU7026. At 96 h after IR-exposure, MTT cell proliferation assays were performed to assess cell viability (n = 4 for each cell line). Radiosensitizing effects were estimated by calculating combination indices (CI) according to Chou and Talalay [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145744#pone.0145744.ref040" target="_blank">40</a>], given on the Y-axis. CI > 1.1 is antagonistic (dark grey), 1.1 ≥ CI ≥ 0.9 is additive (white) and CI < 0.9 is synergistic (light grey). Horizontal lines between the symbols represent the mean combined effect obtained for all neuroblastoma- or fibroblast cell lines. (B) Western Blot analysis of DNA-PKcs protein levels in neuroblastoma cell lines NGP, SHSY5Y, SHEP2, SJNB12, LAN5 and SKNBE(2) and fibroblast cell lines F1366, F1641, F0577 and F2112. β-actin protein levels were used as loading control.</p

Combination treatment with NU7026 and IR induces apoptosis in NGP cells.

<p>(A) Effects of NU7026 plus IR combination therapy versus monotherapy on the cell cycle status of NGP cells. For combination treatment, cells were pre-treated with 10 μM NU7026 for 1 h before exposure to 0.63 Gy IR. Samples were analyzed by FACS analysis at 96 h after treatment. (B) FACS analysis of the effects of combination therapy versus monotherapy on the sub-G1 fraction of cells at 48, 72 and 96 h after treatment. (C) Western Blot detection of total DNA-PKcs and activated DNA-PKcs (via phosphorylation at serine 2056) levels and PARP cleavage after combination therapy versus monotherapy with 10 μM NU7026 and/or 0.63 Gy IR, at 1 h and 96 h after IR-exposure, respectively. β-actin protein levels were used as loading control.</p

Enhanced <i>PRKDC</i> expression in neuroblastoma patients correlates with a poor prognosis.

<p>(A) Comparison between the average Affymetrix <i>PRKDC</i> mRNA expression levels in two independent neuroblastoma datasets (dark grey) versus expression levels in a compiled library of normal tissues and in nonmalignant adrenal glands (light grey). (B) Affymetrix <i>PRKDC</i> mRNA expression in neuroblastoma of 88 individual patients, ordered by the ²log fold transformed level of <i>PRKDC</i>. Color coded tracks below the YY plot give information about neuroblastoma stage (INSS; green = stage 1, dark green = stage 2, light brown = stage 3, blue = stage 4s and red = stage 4), patient survival (green = yes and red = no) and the presence of <i>MYCN</i> amplification and <i>ALK</i> mutation (green = yes and red = no). (C) Kaplan-Meier curve of the survival of neuroblastoma patients with an Affymetrix <i>PRKDC</i> mRNA expression above 656 (n = 8) versus neuroblastoma patients with an Affymetrix <i>PRKDC</i> mRNA expression below 656 (n = 80). The cutoff of 656 was determined using the Kaplan scanner tool in the R2 web application (<a href="http://r2.amc.nl/" target="_blank">http://r2.amc.nl</a>). Samples were sorted according to the expression of <i>PRKDC</i> and divided into two groups on the basis of a cutoff expression value. All cutoff expression levels and the resulting groups were analyzed for survival, with the provision that each group included at least eight samples. For each cutoff level and grouping, the log-rank significance of projected survival was calculated. The graph depicts the best <i>p</i> value corrected for multiple testing (Bonferroni correction).</p

DNA-PKcs inhibitor NU7026 radiosensitizes NGP cells to low IR doses.

<p>(A) Cell viability of NGP cells after co-treatment with NU7026 and IR. Cells were treated with 0, 2, 10 or 20 μM NU7026, 1 h prior to exposure to up to 6.25 Gy IR. At 48, 72, 96 and 120 h after IR-exposure, MTT cell proliferation assays were performed to study inhibitory effects on cell viability. Data represent the mean (n = 4) +/- SD. (B) Cell viability of NGP cells after treatment with 10 μM NU7026 and/or 0.63 Gy IR at 96 and 120 h after IR-exposure. Data represent the mean (n = 4) +/- SD. Statistical differences between untreated and treated NGP cells are indicated as * (<i>p</i><0.05). (C) Effects of NU7026 plus IR combination therapy versus monotherapy on the colony forming capacity of NGP cells. Cells in 0.4% agar in culture medium were seeded on top of a hardened 0.5% agar base layer. After overnight incubation at normal culture conditions, cells were 1 h pre-incubated with 0, 2, 10 or 20 μM NU7026 in 0.4% agar in culture medium before exposure to 0 or 0.63 Gy IR. The following 3 weeks, fresh DMSO or NU7026 in 0.4% agar in culture medium was added to the cells once a week. Colonies were subsequently visualized by 4 h incubation with MTT and counted. The number of colonies formed by untreated NGP cells has been set at 100%. Data represent the mean (n = 3) +/- SD.</p

Newly-derived neuroblastoma cell lines propagated in serum-free media recapitulate the genotype and phenotype of primary neuroblastoma tumours

Recently protocols have been devised for the culturing of cell lines from fresh tumours under serum-free conditions in defined neural stem cell medium. These cells, frequently called tumour initiating cells (TICs) closely retained characteristics of the tumours of origin. We report the isolation of eight newly-derived neuroblastoma TICs from six primary neuroblastoma tumours and two bone marrow metastases. The primary tumours from which these TICs were generated have previously been fully typed by whole genome sequencing (WGS). Array comparative genomic hybridisation (aCGH) analysis showed that TIC lines retained essential characteristics of the primary tumours and exhibited typical neuroblastoma chromosomal aberrations such as MYCN amplification, gain of chromosome 17q and deletion of 1p36. Protein analysis showed expression for neuroblastoma markers MYCN, NCAM, CHGA, DBH and TH while haematopoietic markers CD19 and CD11b were absent. We analysed the growth characteristics and confirmed tumour-forming potential using sphere-forming assays, subcutaneous and orthotopic injection of these cells into immune-compromised mice. Affymetrix mRNA expression profiling of TIC line xenografts showed an expression pattern more closely mimicking primary tumours compared to xenografts from classical cell lines. This establishes that these neuroblastoma TICs cultured under serum-free conditions are relevant and useful neuroblastoma tumour model