Abstract 691: DFMO synergizes with BET inhibitors targeting ODC and MYCN to impede neuroblastoma cell proliferation and tumor initiation
Authors: DeCou Sarah , Zhao Ping , Avequin Tracey , Nagulapally Abhinav , Bond Jeffrey , Sholler Giselle Saulnier 
Published: July 2017

Background: Neuroblastoma (NB) is the most common extracranial solid pediatric tumor and is associated with MYCN amplification. MYCN is a regulator of ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine biosynthesis. Inhibition of this pathway in MYCN-amplified NB tumors has been shown to be a target for treatment. Alpha-difluoromethylornithine (DFMO) inhibits ODC and is currently being used in a Phase II clinical trial for NB. BET inhibitors JQ1 and OTX-015 have been shown to be effective against MYCN-amplified cancers; it is hypothesized that they downregulate MYCN as well as cancer stem cell (CSC) signaling. We propose that inhibiting MYCN with a BET inhibitor, coupled with inhibition of ODC with DFMO, will result in enhanced inhibition of NB growth and tumor-initiating properties.

Methods: Single and combination drug treatments were conducted on BE(2)-C, SMS-KCNR, CHLA90, and one patient-derived cell line. CellTiter-Glo Luminescent Cell Viability Assay was used to determine cell viability in 96-well plates. IC50 values were calculated with a four-parameter variable-slope dose response curve using GraphPad Prism v.5 software. Drug combination studies were conducted in MYCN amplified tumors BE(2)-C and SMS-KCNR using ray designs to evaluate for synergism. IncuCyte ZOOM Live-Cell Imaging system was used for kinetic monitoring of cytotoxicity and apoptosis in NB cells. Western blots measured protein levels of apoptosis markers (cleaved caspase 3 and cleaved PARP) and CSC markers (Nanog, Sox2, NF-kB, CXCR4, Lin28B, and MYCN). Neurosphere assays were used to evaluate tumor initiation via sphere formation.

Results: Cell viability of MYCN amplified NB showed an IC50 range of 1.48-1.69 μM for JQ1 and 839.3 nM-2.36 μM for OTX-015; MYCN non-amplified NB showed an IC50 range of 4.22-11.58 μM for JQ1 and 2.99-11.03 uM for OTX-015. Combination treatment in MYCN NB had a synergistic effect, based on Loewe-additivity as the null hypothesis, for cell viability suppression by ray design experiments for BE(2)-C and SMS-KCNR. Western blots showed greater expression of apoptosis markers and decreased expression of CSC markers in combination relative to single drug treatments. A greater than 50% decrease in neurosphere formation with combination treatment in BE(2)-C, SMS-KCNR, and the patient cell line provides evidence for reduced CSC activity. IncuCyte imaging showed an increase in cell death with time post-treatment.

Conclusion: The combination of DFMO with BET inhibitors has a synergistic effect in treating MYCN amplified NBs as shown by a decrease in cell viability. This combination targets CSC pathways and decreases tumor-initiating ability. Given the lack of effective treatment options for children with relapsed or refractory high-risk NB, this combination may be a promising novel therapy.

Citation Format: Sarah DeCou, Ping Zhao, Tracey Avequin, Abhinav Nagulapally, Jeffrey Bond, Giselle Saulnier Sholler. DFMO synergizes with BET inhibitors targeting ODC and MYCN to impede neuroblastoma cell proliferation and tumor initiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 691. doi:10.1158/1538-7445.AM2017-691