Abstract
Abstract
The AKT pathway is frequently activated in cancer cells and is a defined target for therapeutic intervention.
Methods: RNA expression levels of candidate AKT pathway genes (AREG, EGFR, AKT, mTOR, GSK3α/β) were defined following RNAseq of replicate primary cell cultures (GUMC220 and GUMC221), which were established independently from two areas of the same low-grade salivary mucoepidermoid carcinoma (MEC). Protein expression and phosphorylation levels of the corresponding gene products were characterized using immunohistochemistry on FFPE sections from the original patient tumor, cell pellets, and cell line generated patient-derived xenografts and through western blotting of proteins extracted from cells grown under different culture conditions before and after treatment with AKT inhibitors GSK690693 (ATP-competitive) and MK-2206 2HCl (allosteric inhibitor). The effects of GSK690693 and MK-2206 2HCl on cell viability were tested at different concentrations ranging from 1.2-40μM in 2D and 3D conditions with three different culture media (Conditional media+ ROCK kinase inhibitor Y27632, conditional media without Y27632, and EpiCult-C BasalTM) using CellTiter-GloTM. Doxorubicin was used as a comparative control for evaluating the impact of a chemotherapeutic on cell viability and DMSO as a negative vehicle control. MDA-MB-453 cells were tested as a positive control for MK-2206 2HCl and GSK690693 sensitivity.
Results: Replicate primary cell cultures showed the same chemosensitivity results. MK-2206 2HCl induced a significantly greater reduction in cell viability as compared to GSK690693 in the MEC primary cells. This differential response was replicated in all three 2D culture medias tested (GUMC220_CMY p= 0.0017, GUMC220_CM p=0.0004, GUMC220_EpiC p<0.0001, GUMC221_CMY p=0.0010, GUMC221_CM p<0.0001, and GUMC221_EpiC p=0.0041, One-way ANOVA) with the same pattern reproduced in 3D cultures. Expected changes in levels of p-AKT(Ser473), GSK3α/β, p-GSK3α/β, and p-mTOR were found following treatment with both drugs as compared to vehicle controls. In MK-2206 2HCl treated cells, expression levels were all reduced whereas in GSK690693 treated cells, p-AKT(Ser473), p-AKT(Thr308) were increased and GSK3α/β, p-GSK3α/β, p-mTOR reduced.
Conclusion: Characterization of primary cell cultures using RNAseq led to the identification of a potentially druggable pathway. Direct testing revealed a differential response to the two AKT inhibitors evaluated. Although the two primary cell cultures were established from two geographically distinct areas of the same tumor, response to drug testing was the same and reproducible across different culture modalities. Studies establish the feasibility of deriving primary cells for drug testing and personalized therapy, even from small tumors.
Citation Format: Ahmad M. Alamri, Priscilla A. Furth. A significant differential sensitivity to AKT inhibitors GSK690693 and MK-2206 2HI was identified in primary cells established from a low-grade mucoepidermoid carcinoma [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 2729. doi:10.1158/1538-7445.AM2017-2729