Abstract 3952: Noncoding elements within <i>MYCN</i> mRNA are autonomous drivers of oncogenesis in neuroblastoma

Abstract Neuroblastoma (NB) is a highly metastatic pediatric cancer originating from neural crest cells. Progression is frequently driven by MYCN amplified (MA) disease, a marker of poor prognosis presented in approximately 20% of cases. While MA NB is associated with increased MYCN protein expression, recent results suggest that non-coding RNA (ncRNA) elements within the MYCN mRNA independently contribute to its oncogenic potential. The let-7 microRNA (miRNA) family, which targets the 3′ UTR of MYCN, is sequestered by MYCN mRNA itself in MA disease, thus impairing its function and establishing that ncRNA elements can contribute to disease pathology. To further explore the role of these ncRNA elements, we engineered cells expressing either MYCN-ORF (MYCN protein-coding region only), MYCN-GL (full-length MYCN mRNA from the intact genetic locus), and Null-GL (where EGFP replaces MYCN protein coding sequence but retains all ncRNA elements). In vitro growth analyses showed that MYCN-GL-expressing cells exhibited the most robust growth, followed by Null-GL and MYCN-ORF. In vivo, the Null-GL construct induced the highest incidence and largest tumors of all constructs, followed by MYCN-GL and then MYCN-ORF. Next-generation sequencing revealed that MYCN-GL and Null-GL cells were enriched for targets of microRNAs that also target MYCN, including let-7, miR-101, and miR-34a. This suggests that the MYCN 3’UTR may interact with these microRNAs, thus protecting other mRNA targets. Additionally, MYCN-GL and Null-GL constructs exhibited more dynamic differential gene expression than MYCN-ORF and EGFP-expressing cells. Gene ontology analysis showed that MYCN-GL and Null-GL cells were more enriched in cancer-related pathways, such as RNA metabolism and microRNA processing. Non-coding MYCN mRNA elements may thus regulate broader cellular functions that enhance oncogenesis. Exon usage analysis revealed that MYCN-GL and Null-GL constructs influenced splicing patterns, with increased exon inclusion and exclusion events observed across multiple chromosomes. KEGG pathway analysis of differentially spliced genes further confirmed non-coding RNA element influence on critical oncogenic pathways, including PI3K signaling, TNF signaling, viral carcinogenesis, cell survival, proliferation, and immune evasion. Whole genome sequencing revealed significant similarities in copy number variation (CNV) patterns between MYCN-GL and Null-GL constructs. These results demonstrate that non-coding elements within MYCN mRNA are potent drivers of tumorigenesis, influencing microRNA interactions, splicing patterns, genetic patterning, and overall activation of oncogenic pathways. Our findings provide new insights into the oncogenic mechanisms of MA NB, with broad implications for the genetic contribution of noncoding RNA elements in other oncogene-driven human malignancies. Citation Format: Vishwaben Patel, Lorraine-Rana E. Benhamou, Dr. John T. Powers. Noncoding elements within MYCN mRNA are autonomous drivers of oncogenesis in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3952.