Abstract 500: Bortezomib, a proteasome inhibitor, synergizes with DFMO to inhibit neuroblastoma cell proliferation via the reversal of the LIN28/Let-7 axis
Authors: Rich Maria , Zhao Ping , Nagulapally Abhinav , Bond Jeffrey , Sholler Giselle 
Published: August 2015

Background: The most common extracranial solid pediatric tumor, Neuroblastoma (NB) is responsible for 8-10% of all cancer in children and 15% of pediatric cancer deaths. Due to a high rate of tumor relapse, less than 40% of patients with high-risk disease achieve long-term survival. High incidence of metastatic disease and relapse in NB, as well as cellular heterogeneity of tumor, suggests that high-risk NB is likely due to inherent drug resistance of cancer stem cells (CSCs) that survive treatment in order to regenerate differentiated tumor. One pathway associated with targeting CSCs is the LIN28/Let-7 pathway due to the inductive role of LIN28 in stem cell growth and metabolism. LIN28 is characteristically overexpressed in many malignancies, including NB. Overexpression of LIN28 correlates with poor outcome in NB, therefore drugs that target LIN28/Let-7 axis could be beneficial in treating NB patients by targeting CSCs and preventing relapse of disease. This study proposes the drug combination therapy of Difluoromethylornithine (DFMO)-an inhibitor of ornithine decarboxylase and polyamine biosynthesis-coupled with bortezomib-a proteasome inhibitor-as a new therapeutic upstream target of LIN28/Let-7 pathway and glycolytic metabolism.

Methods: NB cell lines BE(2)-C and SMS-KCNR were used. Cell viability was measured using Calcein AM fluorescent assay at DFMO doses 0.15 - 40 mM, alone and in combination with bortezomib at doses 0.15 - 80 nM. Isobologram cell viability analyses with DFMO in combination with bortezomib were generated using Calcein AM fluorescent assay results. Western blot analysis was used to measure LIN28, MYCN, SOCS3 and NFKB. ATP level per cell was measured using CyQuant fluorescent DNA assay combined with Cell Titer GLO luminescent cell viability assay. Neurosphere assays were used to evaluate sphere formation after DFMO treatment. Xenograft studies were performed with SMS-KCNR cells implanted in the flank of mice and treated with vehicle, 2% DFMO in drinking water, bortezomib 0.5mg/kg IP twice a week, or combination of DFMO and bortezomib.

Results: Cell proliferation assays and isobologram experiments suggest synergistic cytotoxicity of the drug combination in NB cell lines BE(2)-C and SMS-KCNR. Further, western blot analysis and ATP per cell experiments demonstrate the inhibition of overexpressed proteins in the LIN28/Let-7 pathway and inhibition of ATP/cell activity in NB cells. DFMO inhibits neurosphere formation in NB. Xenograft studies show decrease in tumor size with combination treatment.

Conclusion: This study indicates that bortezomib synergizes with DFMO in inhibiting NB cell proliferation and neurosphere formation both in vitro and in vivo. Given the current lack of effective treatments and the high incidence of relapse and metastatic disease for patients, this drug combination offers a potential new therapeutic treatment for children with NB.

Citation Format: Maria Rich, Ping Zhao, Abhinav Nagulapally, Jeffrey Bond, Giselle Sholler. Bortezomib, a proteasome inhibitor, synergizes with DFMO to inhibit neuroblastoma cell proliferation via the reversal of the LIN28/Let-7 axis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 500. doi:10.1158/1538-7445.AM2015-500