Background: Neuroblastoma (NB) is the most common extracranial solid tumor in infants and children, with amplification of the oncogene MYCN being a hallmark of high-risk disease and poor prognosis. Most NB tumors initially respond to chemotherapy; however, over half of these cases relapse, resulting in chemoresistant disease. In this stage of NB there is evidence of p53 inactivation, which suggests a role for p53 in chemo-sensitivity. Although highly mutated in other cancer types, p53 mutations in NB are rare. Previous data have shown that amplification or deregulation of MDM2, a negative regulator of p53, may be the more common mechanism of p53 suppression and chemoresistance in NB. MYCN and MDM2 have been shown to interact and contribute to NB growth and disease progression. In vitro treatment of NB cells with MDM2 inhibitors has shown promise in increasing the expression of p53, leading to a decrease in proliferation, and increasing apoptosis. BET (Bromodomain and Extra-Terminal domain) inhibitors have also been shown to be effective in treating NB cells in vitro, decreasing MYCN expression, and resulting in increased apoptosis and differentiation. We hypothesize that the combination treatment of a MDM2 inhibitor (CGM097) with a BET inhibitor (OTX015) will result in greater p53 activation and a subsequent synergistic increase in NB cell death.
Methods: Single-agent IC50 values for both CGM097 and OTX015 were obtained for the established NB cell lines SMS-KCNR, SH-SY5Y, BE(2)-C, and CHLA-90 using Calcein AM and Cell Titer-Glo cell viability assays after 48 hours of treatment. Drug combination studies were performed to evaluate synergism between CGM097 and OTX015 in the treatment of NB cells. IncuCyte ZOOM live cell imaging was used for kinetic monitoring of apoptosis with single-agent and combination drug treatment in NB cells. Western blot analysis was used to determine protein levels of apoptosis (Caspase-3) and MYCN/p53 pathway targets (MYCN, MDM2 and p53).
Results: IC50 values for the four established NB cell lines ranged from 0.384 µM-13.5 µM and 7.25 µM-42.79 µM for CGM097 and OTX015, respectively. The combination treatment exhibited synergistic effects in wild-type p53 NB cells, with supporting data obtained from IncuCyte imaging tracking real-time cell death. IncuCyte showed induction of caspase-mediated apoptosis confirmed by Western blot analysis of Cleaved Caspase 3. Pathway analysis shows the importance of wild-type p53 for treatment effect.
Conclusion: This study indicates that the combination of CGM097 and OTX015 synergistically decreases viability in NB cells with wild-type p53 expression. Further in vitro and in vivo work will be necessary to elucidate the mechanisms behind this interaction and in vivo efficacy.
Citation Format: Tyler P. Maser, Joseph W. Zagorski, Austin J. Goodyke, Elizabeth A. VanSickle, Jeffrey P. Bond, Giselle L. Saulnier Sholler. The MDM2 inhibitor CGM097 synergizes with the BET inhibitor OTX015 to induce cell death in neuroblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3191.