S. Chukkapalli1, N. Neamati2, T. Thomas1, V. Castle3, E. Newman1 1University Of Michigan,Surgery,Ann Arbor, MI, USA 2University Of Michigan,Medicinal Chemistry,Ann Arbor, MI, USA 3University Of Michigan,Pediatrics,Ann Arbor, MI, USA
Introduction: Neuroblastoma (NB) is the most common extracranial solid tumor in children, arising from neural crest stem cells. Despite multimodality therapies, the 5-year survival rate of patients with high-risk tumors is less than 50%. Previously, we found that tumorigenic and high-risk NB cells are defective in DNA double-strand break (DSB) repair, with elevated expression of alternate NHEJ (alt-NHEJ) pathway components, compared to classical NHEJ. We have shown that inhibiting DNA Ligase III (Lig3) and Ligase I (Lig1) lead to DSB accumulation and cell death. We hypothesize that inhibiting alt-NHEJ components by a novel alt-NHEJ ligase inhibitor (CH43) may sensitize NB cells to standard chemotherapeutic drugs.
Methods: To test efficacy of a novel Lig3 inhibitor (CH43) on NB cells, a panel of stromal (SH-EP1), and tumorigenic (IMR32, SH-SY5Y) NB cell lines were cultured and treated with CH43 for 24, 48 and 72 hours. At end-point, cell viability analysis was performed with CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (MTS) and IC50 values were calculated. In order to measure the DNA damage response due to Lig3 inhibition, CH43 treated cells were immunostained with γH2AX (a marker for DSB) followed by quantification of γH2AX foci in the nuclei of treated cells. Protein lysates were analyzed by immunoblot to assess the extent of Lig3 inhibition in CH43 treated cells. To evaluate for synergistic activity between CH43 and doxorubicin, NB cells were treated with CH43 for 24 hours, followed by doxorubicin treatment for 48 hours. Cell viability was assessed by the MTS assay, and combination index values were calculated with Compusyn Software.
Results: Each NB cell type was sensitive to CH43 in a dose-dependent manner, with IC50 values ranging from 2uM to 20uM. Immunoblotting analysis of protein lysates collected from CH43 treated cells confirmed inhibition of Lig3 and Lig1 in tumorigenic NB cells. We also noted increased expression of DNA Ligase IV, a classical NHEJ pathway component in these cells. Quantification of γH2AX foci in the nucleus of CH43 treated cells showed that inhibition of Lig3 with CH43 induced DSBs in NB cells. NB cells pretreated with CH43 were more sensitive to Doxorubicin compared to cells without pretreatment; suggesting CH43 and doxorubicin exhibit strong synergism against tumorigenic NB cell lines.
Conclusion: Collectively, these findings strongly suggest that sensitizing tumorigenic NB cells to anti-cancer drugs by inhibiting alt-NHEJ components is an effective therapeutic approach to treat high-risk NB.
