Abstract 7038: Direct targeting of DNMT1 inhibits high-risk neuroblastoma growth through epigenetic reprogramming

Abstract DNA Methyltransferase 1 (DNMT1) mediated hypermethylation represents a crucial epigenetic modification implicated in neuroblastoma (NB), a devastating pediatric cancer with a 60% relapse rate. DNMT1, a prominent member of the DNA methyltransferase family of enzymes, catalyzes the transfer of methyl groups to the 5-carbon position of cytosine residues within DNA sequences. While extensive research has established that hypermethylation of CpG islands leads to transcriptional silencing of tumor suppressor genes across various cancer types, the specific molecular mechanisms and therapeutic implications of DNMT1-mediated hypermethylation in NB remained largely unexplored. In this study, we analyzed the publicly available R2 genomic dataset, which revealed the direct correlation between DNMT1 overexpression and decreased overall NB patient survival rates. Further, we performed a small molecule screening and identified a highly potent and selective DNMT1 inhibitor. Therapeutic targeting of DNMT1 with this inhibitor resulted in significant dose-dependent reductions in multiple hallmarks of cancer progression, including overall cell proliferation, colony formation capacity, and 3D spheroid development. Furthermore, the DNMT1 inhibitor induced cell cycle arrest in the G2/M phase checkpoint and significantly enhanced apoptotic cell death in NB cells. These promising in vitro findings were validated through in vivo tumor xenograft studies, where the DNMT1 inhibitor significantly inhibited NB tumor growth without any observable toxicity in preclinical models. Further, we employed MethylFlash technology to quantify 5-methylcytosine (5-mc) levels, which verified a significant reduction (p < 0.0001) in global DNA methylation status following exposure to increasing concentrations (5, 10, and 20 µM) of the DNMT1 inhibitor. Further molecular analyses revealed profound effects on vital gene and protein expression patterns alongside an enhancement of NB cell sensitivity to conventional chemotherapeutic agents. These findings collectively establish DNMT1 inhibition as a promising therapeutic strategy for NB patients. Our data demonstrates the role of DNMT1-mediated hypermethylation in NB tumorigenesis while establishing that targeted DNMT1 inhibition effectively reduces hypermethylation, thereby suppressing malignant proliferation, inhibiting colony formation, and preventing both 3D spheroid development and in vivo tumor growth. Our data also highlights that the direct DNMT1 targeting approach is a promising epigenetic-based approach for NB treatment. Further mechanistic research of DNMT1 inhibition in NB progression is essential to facilitate the clinical translation of this promising therapeutic strategy. Citation Format: Danielle C. Rouse, Saurabh Agarwal. Direct targeting of DNMT1 inhibits high-risk neuroblastoma growth through epigenetic reprogramming [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 7038.