Abstract 7047: Impact of MYCN inhibition on lineage state and GD2 expression in neuroblastoma

Abstract Neuroblastoma (NB) is a malignant cancer that originates in the developing peripheral nervous system and accounts for ∼15% of all childhood cancer deaths. Some NBs are easily cured, but those classified as high risk require treatment that includes chemotherapy, surgery, autologous stem cell transplant, radiation, and immunotherapy with antibodies targeting the disialoganglioside GD2. Despite this extensive therapy, only ∼50% of children survive. Even in these high-risk cases, most of the NB cells respond to initial therapy, but a subset of cells survives (persister cells) and grows back into resistant relapsed disease. Two important factors in relapse are MYCN amplification and intratumoral heterogeneity. MYCN, a transcription factor of the MYC family, is amplified in about 40% of high-risk NB cases and is associated with a particularly poor prognosis. Intratumoral heterogeneity is also thought to contribute to relapse. NB cells can exist in two different lineage states: a chemosensitive adrenergic (ADRN) state and chemoresistant mesenchymal (MES) state. With the development of improved pan-MYC inhibitors and the advancement of MYC-targeting therapies into clinical trials, there is a new need and opportunity to investigate how NB responds to MYCN inhibition, how this impacts lineage state, and how this changes GD2 expression. We investigated the effects of MYCN inhibition in MYCN-amplified ADRN cell lines using the small molecule pan-MYC inhibitor MYCi975. Cell viability assays, western blot analysis, quantitative PCR, and flow cytometry analysis were conducted to evaluate the impact of MYCN inhibition. We found that treatment with MYCi975 results in a rapid decrease in the protein levels of MYCN, MYCN target genes, and ADRN state markers, but not in new expression of MES state markers or changes in the expression of the immunotherapeutic target GD2. Doses that effectively decrease MYCN result in a loss of cell viability after 3-5 days. However, rare cells survive prolonged treatment (persister cells) and are able to resume proliferation once MYCi975 is removed. These recovered cells demonstrate relative resistance to retreatment with MYCi975 with a ∼2-fold increase in the IC50 compared to naïve cells. Additionally, these resistant cells recover basal levels of MYCN and ADRN markers but have decreased surface GD2. Our findings support the likelihood that some NB cells will survive therapies that inhibit MYCN and provide a model to investigate the identity and potential vulnerabilities of these cells. Furthermore, we find a role for MYCN in maintaining the ADRN lineage state but not in maintaining GD2 expression. However, GD2 is downregulated in resistant cells with implications for the relative timing of MYCN-targeting and GD2 targeting therapies. Ongoing work will be presented on transcriptomic changes after MYCi975 treatment and in resistant cells. Citation Format: Angelle L. Brown, Sridhar Veluvolu, Matthew Shapiro, Connor Hennessey, Adam Wolpaw. Impact of MYCN inhibition on lineage state and GD2 expression 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 7047.