Abstract 1489: Targeting the DNA double-strand break repair pathways in MYCN-amplified neuroblastoma increases tumor sensitivity to chemotherapy
Authors: Mahnaz Norouzi, Subin Kim, B. Mark Evers, Eric J. Rellinger, Piotr Rychahou
Journal: Cancer Research (2025)

Abstract Background: DNA double-strand break (DSB) repair is regulated by non-homologous end joining (NHEJ) and homologous recombination (HR). In neuroblastoma (NB), extensive genomic instabilities such as MYCN amplification, 11q and 1p deletions, and 17q gain are linked to tumor progression and high-risk disease. These abnormalities are associated with defective DSB repair and are enriched in high-risk NB. Here we demonstrate the association between DSB repair pathways and MYCN amplification (MNA) in high-risk NB and explore the potential response of MNA NB to therapies targeting these pathways. Methods: The R2 Genomics Analysis and Visualization Platform was used for gene set enrichment (GSEA) and differential expression analysis in NB cohorts. DNA-PKcs gene knockdown models, along with Peposertib and Olaparib treatment, were used to inhibit DNA repair activity. Western blotting (WB) was performed to verify DNA-PKcs inhibition and measure PARP cleavage in response to DNA-PKcs inhibition in BE(2)-C and SK-N-DZ cells. DNA damage was induced by Doxorubicin and detected using γ-H2AX immunofluorescence staining followed by confocal microscopy. Clonogenic assays were performed to assess the efficacy of DNA repair inhibition on the survival of NB cells. Results: GSEA using the Kocak cohort study, which contains microarray gene expression data from 649 NB samples from the NB Research Consortium (NRC), revealed that NHEJ was upregulated in MNA high-risk NB (GSE score = 1.183), while the HR pathway showed moderate downregulation (GSE score = -0.428). Additionally, differential analysis revealed that key components of NHEJ, including and PRKDC, XRCC5, NHEJ1 and Rad50, showed significantly higher expression in MNA high-risk NB compared to non-MNA (p &amp;lt; 0.05). Confocal imaging demonstrated increased γ-H2AX foci formation in DNA-PKcs knockdown cells, indicating enhanced DSBs in response to DNA damage due to the loss of active NHEJ repair. Increased cleaved PARP was demonstrated in DNA-PKcs siRNA SK-N-DZ and BE(2)-C knockdown cells, indicating increased apoptosis in response to its inhibition in MNA NB cells. Clonogenic analysis showed a significant decrease in NB cell survival in response to Doxorubicin treatment, with a more pronounced decrease in response to the combination with both Peposertib and Olaparib. Conclusion: High-risk NB is correlated with extensive genomic instability, and here we showed that upregulation of NHEJ is associated MNA high-risk NB. DNA-PKcs knockdown studies revealed that the survival of MNA NB cells depends on the activity of NHEJ pathway, paving the way for more targeted treatments. The significant response of NB cells to the combination of Peposertib and Olaparib highlights a promising strategy to enhance NB cell sensitivity to conventional therapies, particularly Doxorubicin. Citation Format: Mahnaz Norouzi, Subin Kim, B. Mark Evers, Eric J. Rellinger, Piotr Rychahou. Targeting the DNA double-strand break repair pathways in MYCN-amplified neuroblastoma increases tumor sensitivity to chemotherapy [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 1489.