Abstract 5721: The super-enhancer driven long noncoding RNA lncNB promotes neuroblastoma tumorigenesis
Authors: Mondal Sujanna , Wong Matthew , Tee Andrew , Ho Nicholas , Ziegler David S. , Marshall Glenn M. , Liu Pei Y. , Dinger Marcel , Liu Tao 
Published: September 2020

Background: While MYCN-amplified neuroblastoma has been the focus of neuroblastoma research in the past three decades, most human neuroblastomas do not harbour MYCN oncogene amplification, and their tumorigenic factors are unknown. The expression of critical oncogenes is driven by transcriptional super-enhancers which are controlled by the BET bromodomain protein BRD4 and the transcription factor TFIIH-associated kinase CDK7. Long noncoding RNAs (lncRNAs) play important roles in cancer.

Aims: To determine the mechanism through which the novel lncRNA lncNB is up-regulated in MYCN-non-amplified neuroblastoma; to determine the role of lncNB in neuroblastoma cell proliferation, survival and tumour progression; and to identify the mechanism through which lncNB exerts oncogenic effects.

Methods and results: RNA sequencing analysis identified lncNB as one of the transcripts most dramatically over-expressed in MYCN non-amplified neuroblastoma cell lines and tumour tissues, and Kaplan Meier analysis showed that high levels of lncNB expression in neuroblastoma tissues correlated with poor prognosis in 476 patients. Examination of published chromatin immunoprecipitation sequencing data revealed super-enhancers at the lncNB gene locus only in MYCN non-amplified neuroblastoma cells.

RT-PCR experiments showed that transfection with two independent BRD4 siRNAs or treatment with the BRD4 inhibitor AZD5153 or the CDK7 inhibitor THZ1 considerably reduced lncNB expression and cell proliferation in MYCN non-amplified neuroblastoma cells. Alamar blue assays, cell cycle analysis, BRDU assay and clonogenic assays revealed that lncNB knockdown with siRNAs or doxycycline-inducible shRNAs in MYCN non-amplified neuroblastoma cells led to growth inhibition, apoptosis and dramatic reduction in clonogenic capacity. RNA-binding protein pull-down and RNA immunoprecipitation assays identified MSI as a binding partner for the lncNB lncRNA; and RNA sequencing has identified BMX as the downstream target of lncNB. In addition, Balb/C nude mice were xenografted with doxycycline-inducible lncNB shRNA MYCN non-amplified neuroblastoma cells. Knocking down lncNB with doxycycline in food considerably suppressed tumour progression and improved mouse survival.

Conclusions: The data suggest that the lncRNA lncNB is upregulated by super-enhancer activity in MYCN non-amplified neuroblastoma cells, that lncNB up-regulation induces neuroblastoma cell proliferation, survival, clonogenic capacity and tumorigenesis, and that targeting lncNB expression can be an effective therapeutic strategy.

Citation Format: Sujanna Mondal, Matthew Wong, Andrew Tee, Nicholas Ho, David S. Ziegler, Glenn M. Marshall, Pei Y. Liu, Marcel Dinger, Tao Liu. The super-enhancer driven long noncoding RNA lncNB promotes neuroblastoma tumorigenesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5721.