Background: Neuroblastoma (NB) is associated with MYCN oncogene amplification occurring in approximately 30% of NBs and is associated with poor prognosis. MYCN is linked to a number of genes including ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. ODC expression is elevated in NB. Alpha-difluoromethylornithine (DFMO), an ODC inhibitor, is currently being used in a Phase I clinical trial for treatment of NB. However, cancer cells treated with DFMO may overcome their polyamine depletion by the uptake of polyamines from extracellular sources. A novel polyamine transport inhibitor, AMXT-1501, has not yet been tested on NB. We proposed that inhibiting ODC with DFMO, coupled with polyamine transport inhibition by AMXT-1501, would result in increased NB growth inhibition.
Methods: Single and combination drug treatments were conducted on three NB cell lines in vitro and in a xenograft mouse model in vivo. Cell viability was measured using Calcein AM fluorescent assay. Real-time cell proliferation was measured using the xCELLigence system. Western blot analysis was used to measure cleaved and full PARP, cleaved and full caspase 3, phosphorylated Rb and MYCN levels. ATP level per cell was measured using CyQuant fluorescent DNA assay combined with the Cell Titer GLO luminescent cell viability assay. Reverse-phase HPLC was used to measure polyamine levels in cells.
Results: DFMO IC50 values ranged from 20.76 to 33.3 mM, and AMXT-1501 IC50 values ranged from 14.13 to 17.72 μM in NB cell lines. Low dose combination treatment (2.5 μM AMXT-1501, 2.5 mM DFMO) synergistically inhibited cell viability and proliferation in vitro. Combination treatment decreased MYCN expression and resulted in hypophosphorylation of pRb, suggesting cell growth inhibition. Increased expression of cleaved PARP and cleaved caspase 3 in combination-treated cells starting at 48 hours suggested apoptosis. The combination treatment resulted in intracellular polyamine pool depletion and decreased intracellular ATP. The combination treatment also significantly reduced tumor size in NB xenograft mice in vivo.
Conclusion: Given the current lack of effective therapies for relapsed/refractory NB patients and the preclinical effectiveness of DFMO with AMXT-1501, this combination treatment provides promising preclinical results. DFMO and AMXT-1501 may be a potential new therapy for children with NB.
Citation Format: Ann M. Kendzicky, Ping Zhao, Katherine Samal, Lisette P. Yco, Heather McClung, Eugene W. Gerner, André S. Bachmann, Giselle L. Sholler. Targeting both ornithine decarboxylase and polyamine transport inhibits tumor growth in neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2765. doi:10.1158/1538-7445.AM2013-2765