G. J. Bhatt1, A. P. Williams2, L. L. Stafman2, J. M. Aye3, J. E. Stewart2, E. A. Beierle2 3University Of Alabama at Birmingham,Hematology And Oncology,Birmingham, Alabama, USA 1Alabama College of Osteopathic Medicine,Dothan, AL, USA 2University Of Alabama at Birmingham,Surgery,Birmingham, Alabama, USA
Introduction:
Neuroblastoma is the most common pediatric extracranial solid tumor. Originating from neural crest cells, it is often unpredictable in nature. In high risk disease, the 5-year survival rate is only about 40-50%. Given its prevalence in the pediatric population and its lethality, aggressive research is needed to combat this tumor. Much promise has been found in the study of Enhancer of Zeste Homologue 2 (EZH2), a histone methyltransferase. EZH2 inhibitors are currently undergoing clinical trials for the treatment of pediatric solid tumors. EZH2 has been found to affect neuroblastoma cell survival and tumorigenesis. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase which has been proven to decrease survival and proliferation in neuroblastoma1. We have shown that there is a direct interaction between EZH2 and FAK in neuroblastoma. EZH2 inhibition, as well as FAK inhibition, have shown promise in treating many solid tumors, but combination therapy of the two has not been investigated in neuroblastoma. Our objective was to show that combined inhibition of FAK and EZH2 would decrease neuroblastoma survival and proliferation.
Methods:
Long-term passaged SH-EP and WAC2 neuroblastoma cell lines were utilized. Cells were treated with a combination of doses of GSK343 (EZH2 inhibitor) and PF-573, 228 (FAK inhibitor) on 96 well plates. Cell viability and proliferation were measured with alamarBlue® and Cell Titer96® assays, respectively. The Chou-Talalay method was used to analyze results of the combination therapy, and combination indices (CI) were calculated with a combination index less than 1 representing synergism.
Results:
The SH-EP cell line displayed synergistic effects from combination therapy of GSK343 and PF, showing markedly decreased viability after treatment. Treatment with GSK343 alone resulted in an LD50 of 63.5mM, and PF treatment alone resulted in an LD50 of 8.2mM. At a constant dose of 10mM GSK343 or 15mM GSK343, combination with 2, 4, or 6mM PF yielded combination indices of 0.875 and 0.919, respectively. Constant doses of 2 or 4mM PF combined with 5, 10, or 15mM GSK33 yielded combination indices 0.825 and 0.828, respectively. These values, which are less than one represent synergy.
Conclusion:
The SH-EP cell line displayed synergistic effects from combination therapy of GSK343 and PF, showing markedly decreased viability after treatment. In conclusion, these data indicate that further investigation of EZH2 inhibition in combination with FAK inhibition is warranted in neuroblastoma.
