V. Naga1, C. Gordon1, J. Mazar1, T. Westmoreland1 1Nemours Children’s Hospital,Biomedical Research,Orlando, FL, USA
Introduction: MYCN amplification is a prognostic biomarker associated with poor prognosis of neuroblastoma in children, accounting for 40 to 50 percent of all high-risk cases. The overall survival of children with MYCN amplified neuroblastoma has only marginally improved within the last 20 years. The bromodomain inhibitor JQ1 has been shown to downregulate MYCN in sensitive MYCN amplified neuroblastoma cells (Pussaint et al.). We hypothesize that JQ1 treatment will result in a greater decreased viability in MYCN amplified versus MYCN non-amplified neuroblastoma cells, which, in turn, may have therapeutic implications.
Methods: We used three MYCN amplified neuroblastoma lines [IMR-32, SMS-KAN, and SK-N-Be(1)] and three MYCN non-amplified neuroblastoma lines (SK-N-AS, LAN-6, and CHLA-42) to perform MTS cell viability assays and caspase 3/7 apoptosis assays. Cells were treated at four different concentrations (1 µM, 2 µM, 4 µM, and 8 µM) of JQ1 and its ortho-isomer. Cell viability assays were performed by adding MTS reagent 72 hours after JQ1 treatment and measuring the resulting change in absorbance. For the apoptosis assays, cells were treated at 8 µM of JQ1 and its ortho-isomer. After 48 hours of treatment, a caspase 3/7 substrate was added to the cells and changes in luminescence were measured.
Results: After treatment with JQ1, cell viability decreased in a dose-dependent manner in all cell lines tested. The difference in cell viability between the MYCN amplified and MYCN non-amplified cell lines was most pronounced at the 8 µM concentration of JQ1; cell survival was significantly lower in all the MYCN amplified cells compared to MYCN non-amplified cells. However, responses to the ortho-isomer of JQ1 were not consistent with respect to cell viability. An analysis of the changes in caspase 3/7-dependant apoptosis indicated significant increases in both MYCN amplified and MYCN non-amplified cell lines, though MYCN amplified cells appeared to be more sensitive.
Conclusion: MYCN amplified cell lines demonstrated a more pronounced decrease in cell viability after treatment with JQ1 than the MYCN non-amplified cell lines. This is supported by the caspase 3/7-dependant apoptosis data, which showed that MYCN amplified cell lines showed higher rates of apoptosis after JQ1 treatment. The ortho-isomer of JQ1 had a limited effect on the induction of apoptosis but had a variable effect on cell viability, which suggests that these changes in cell viability may induce a form of cell cycle arrest, indicating it is not entirely harmless as previously reported. Regardless, these data support the hypothesis that JQ1 may serve as an effective therapeutic in the treatment of MYCN amplified neuroblastoma by decreasing both cell viability and increasing the rate of cellular apoptosis in these high-risk pediatric cancers.