Epigenetic Targeting of DNMT1 Inhibits Neuroblastoma Growth: anin vitroandin vivostudy

Abstract ID 97834 Poster Board 049 Purpose: Neuroblastoma (NB) is a solid tumor that develops from premature nerve cells of the sympathetic nervous system and is reported as the most common cancer in children younger than 1 year. Despite advanced treatment modalities such as surgery, radiation, and intensive chemotherapies, high-risk NB still only has a survival rate of approximately 50%. These critical statistics mandate the development of novel, less toxic, and more effective therapies for NB. Epigenetic mechanisms like hypermethylation are reported to drive cancer malignancy, metastasis, tumorigenesis, and relapse in multiple cancer types, including NB. Previous studies have reported high DNA methyltransferase 1 (DNMT1) expression in NB patients and associated DNMT1 overexpression with poor overall NB prediction. In the present study, we determine the effect of DMNT1 inhibition on NB growth using both in vitro and in vivo NB models. Our hypothesis is that direct inhibition of DNMT1 with natural DNMT1 inhibitor (nDNMT1i) will inhibit NB proliferation and growth. Methods: R2 Genomic Database platform was used to analyze various NB patient datasets and their correlation with DMNT1. We screened various DNMT1 inhibitors using cell cytotoxicity assays and selected the best hit, a nDNMT1i. To determine the effect of nDNMT1i on NB cell proliferation, we utilized various 2D and 3D assays like cytotoxicity assay, colony formation assay, and 3D spheroid assay. Further, we performed Apoptosis and Cell cycle assays using specific kits and an Attune Flow Cytometer. Western Blot assays were performed using standard methods, and a mouse xenograft model was utilized to determine the effect of nDNMT1i in vivo. Results: Genomic data analysis of 1235 NB patients revealed that expression of DMNT1 is inversely correlated with overall poor survival. DNMT1 expression was also found to be correlated with NB stage progression and tumor MYCN status, highlighting the role of DNMT1 in NB. A DNMT1 inhibitor screening revealed a nDNMT1i as the most efficacious and potent DNA methyltransferase 1 (DNMT1) inhibitor in NB cells. nDNMT1i inhibits cell proliferation in different NB cell lines, including MYCN-amplified and -non-amplified cell lines with IC50 ranging between 5-10μM. nDNMT1i significantly (pin vivo NB xenograft model to determine the efficacy of nDNMT1i and found that nDNMT1i induced necrosis in NB tumors and sensitizes NB tumors to chemotherapy etoposide. We have not observed any toxicities related to nDNMT1i treatments in NB mouse models. Conclusion: Overall, our data highlights the role of DNMT1 and epigenetic regulators in NB development and proliferation. nDNMT1i significantly inhibits NB growth in both in vitro and in vivo tumor models. Our data suggests that direct targeting of DNMT1 is an effective therapeutic approach for NB, and combining these strategies with current chemotherapies is a clinically tractable approach. Future development of these epigenetic-based targeted approaches will provide effective therapies for children battling with NB.