Absence of telomerase reverse transcriptase promoter mutations in neuroblastoma

Maintenance of telomere length is a critical hallmark of malignant transformation. While silenced in somatic cells, telomerase reverse transcriptase (TERT), the catalytic subunit of telomerase, is frequently overexpressed in malignant cells thereby maintaining their telomere length. Specific point mutations in the TERT promoter region have recently been identified in melanoma and other tumor entities resulting in high TERT expression. Neuroblastoma is the most common extracranial tumor of childhood, arising from neural-crest progenitor cells. TERT overexpression has been observed in the majority of neuroblastoma. Taking into consideration that TERT promoter mutations are frequently described in neural-crest-derived tumors such as melanoma, as well as a variety of other neuronal tumors, the present study analyzed the frequency of TERT promoter mutations in primary neuroblastoma and neuroblastoma cell lines. In 131 neuroblastoma primary tumors representing the whole spectrum of neuroblastoma, no TERT promoter mutations were detected. However, in 3 out of 19 neuroblastoma cell lines the previously described C228T TERT promoter mutation was present. In conclusion, the TERT promoter mutations are not a frequent mechanism of TERT overexpression in neuroblastoma

The GSK461364 PLK1 inhibitor exhibits strong antitumoral activity in preclinical neuroblastoma models

Polo-like kinase 1 (PLK1) is a serine/threonine kinase that promotes G2/M-phase transition, is expressed in elevated levels in high-risk neuroblastomas and correlates with unfavorable patient outcome. Recently, we and others have presented PLK1 as a potential drug target for neuroblastoma, and reported that the BI2536 PLK1 inhibitor showed antitumoral actvity in preclinical neuroblastoma models. Here we analyzed the effects of GSK461364, a competitive inhibitor for ATP binding to PLK1, on typical tumorigenic properties of preclinical in vitro and in vivo neuroblastoma models. GSK461364 treatment of neuroblastoma cell lines reduced cell viability and proliferative capacity, caused cell cycle arrest and massively induced apoptosis. These phenotypic consequences were induced by treatment in the low-dose nanomolar range, and were independent of MYCN copy number status. GSK461364 treatment strongly delayed established xenograft tumor growth in nude mice, and significantly increased survival time in the treatment group. These preclinical findings indicate PLK1 inhibitors may be effective for patients with high-risk or relapsed neuroblastomas with upregulated PLK1 and might be considered for entry into early phase clinical trials in pediatric patients

Targeting MYCN-Driven Transcription By BET-Bromodomain Inhibition

Purpose: Targeting BET proteins was previously shown to have specific antitumoral efficacy against MYCN-amplified neuroblastoma. We here assess the therapeutic efficacy of the BET inhibitor, OTX015, in preclinical neuroblastoma models and extend the knowledge on the role of BRD4 in MYCN-driven neuroblastoma. Experimental Design: The efficacy of OTX015 was assessed in in vitro and in vivo models of human and murine MYCN-driven neuroblastoma. To study the effects of BET inhibition in the context of high MYCN levels, MYCN was ectopically expressed in human and murine cells. The effect of OTX015 on BRD4-regulated transcriptional pause release was analyzed using BRD4 and H3K27Ac chromatin immunoprecipitation coupled with DNA sequencing (ChIP-Seq) and gene expression analysis in neuroblastoma cells treated with OTX015 compared with vehicle control. Results: OTX015 showed therapeutic efficacy against preclinical MYCN-driven neuroblastoma models. Similar to previously described BET inhibitors, concurrent MYCN repression was observed in OTX015-treated samples. Ectopic MYCN expression, however, did not abrogate effects of OTX015, indicating that MYCN repression is not the only target of BET proteins in neuroblastoma. When MYCN was ectopically expressed, BET inhibition still disrupted MYCN target gene transcription without affecting MYCN expression. We found that BRD4 binds to super-enhancers and MYCN target genes, and that OTX015 specifically disrupts BRD4 binding and transcription of these genes. Conclusions: We show that OTX015 is effective against mouse and human MYCN-driven tumor models and that BRD4 not only targets MYCN, but specifically occupies MYCN target gene enhancers as well as other genes associated with super-enhancers. (C) 2015 AACR