Effect of extrachromosomal DNA (ecDNA) on <i>MYCN</i> amplified neuroblastoma and patient outcomes.

Abstract

3090 Background: Recurrent cytogenetic abnormalities represent candidate therapeutic targets for children with neuroblastoma (NB). MYCN oncogene amplification is associated with significantly worse survival rates for children with NB and remains one of the primary predictors of patient prognosis. MYCN amplifications in NB can be found both within the linear genome and on circular extrachromosomal DNA (ecDNA), and therapeutic targeting of the mechanisms underlying MYCN amplification represents a novel and promising strategy in NB. However, the molecular features and clinical and biological significance of these amplifications in NB tumors are not sufficiently understood. Methods: Whole genome and RNA sequencing data were analyzed for NB cell lines and NCI TARGET NB samples using AmpliconSuite software for ecDNA identification and characterization. GISTIC was used for identification of recurrently amplified regions. Gene expression levels were determined using StringTie, and gene clustering heatmaps were generated using FeatureCounts software. For differential gene expression analyses, samples were divided into ecDNA + and ecDNA - , and genes contained on ecDNA were compared to the same regions on linear DNA across samples using DESeq2. Associations between ecDNA quantity, content, and patient survival were performed using multivariate Cox regression survival analysis. Associations of gene expression with patient survival were performed using the R2 Platform. The efficacy of targeting ecDNA-associated gene products was assessed using live cell imaging and cell viability assays. Results: WGS analysis confirmed 7/20 NB patient tumors from the TARGET database to be ecDNA amplified with 1-5 independent ecDNA elements and MYCN gene expression correlated with the ecDNA copy number. ecDNAs in MYCN -amplified neuroblastoma cell lines contained distinct gene combinations and possessed unique structures. MYCN overexpression in NB cells has been shown to be associated with replication stress (RS), and tumor cells containing ecDNA are hyper-reliant on the DNA damage response (DDR) kinase CHK1 to manage heightened replication stress. Expression of the CHK1 gene was associated with neuroblastoma patient outcomes and neuroblastoma was most significantly associated with CHK1 RNA dependency. We further validated CHK1i as a promising therapeutic strategy in MYCN amplified NB, as CHK1 inhibition with the novel inhibitor BBI-2779 was most effective against ecDNA+, MYCN -amplified neuroblastoma cell lines. Conclusions: Our results emphasize the critical role of ecDNA in NB. We identify a synthetic lethality axis shaped by ecDNA MYCN amplification and CHK1 dependence. We further demonstrate the feasibility of targeting this vulnerability through CHK1 inhibition, thus offering new avenues for treatment in MYCN amplified tumors.