Identifying targetable markers of resistance to dual TORC1/2 inhibition in endometrial cancer cell lines.

Abstract

e17633 Background: For patients with endometrial cancer, the PI3K/AKT/mTOR pathway is an attractive target due to dysregulation of the pathway secondary to commonly found genetic alterations. Previous trials of mTOR inhibitors in endometrial cancer have shown modest clinical activity and tolerability. Here we present a proteomics based approach to identify novel partners that will enhance the efficacy of dual TORC1/2 inhibition. Methods: Five endometrial cell lines (AN3CA, ECC, HEC1A, HEC1B, RL952) were treated with the TORC1/2 inhibitor TAK228 or vehicle for one hour and protein concentrations were analyzed by reverse phase protein arrays (RPPA). We examined baseline protein and changes in individual proteins after treatment with TAK228 to determine correlations with sensitivity to TAK228. Identified proteins were then targeted with single agent pharmacologic inhibition and cytotoxicity measured by XTT analysis. Target inhibition was validated using WB analysis. Cooperativity in cytotoxicity for relevant combinations was measured using the methods of Chou-Talalay. Other phenotypic endpoints including migration, cell cycle alterations by flow cytometry and cell growth using live cell imaging were also collected. Results: Based on proteomics analysis, altered phosphorylation of CHK1(S296), CHK2(T68) and increasing concentration of VEGFR2 were strongly correlated with TAK228 resistance. TAK228 (TORC1/2, IC50 4-20nM), AZD7762 (CHK1/2, IC50 15-160nM), LY2603618 (CHK1, IC50 260nM-7.5mM) and cediranib (VEGFR2, 800nM-1mM) all had significant cytotoxic effects as individual agents on multiple endometrial cell lines. AZD7762 in combination with TAK228 led to synergism in all cell lines tested (mean combination index, 0.2-0.8). Single agent AZD7762 increased the number of cells in S-Phase and decreased cellular migration. Addition of recombinant VEGF-A to cell culture enhanced resistance to TAK228 in a dose dependent manner as measured by cell growth assays using live cell imaging. Combination of cediranib and TAK228 also showed significant synergism in multiple cell lines. Conclusions: RPPA identified changes in phosphorylated CHK1/2 and total VEGFR2 as being correlated to resistance to dual TORC1/2 inhibition with TAK228 in multiple endometrial cancer cell lines. While single agent activity of TORC1/2, CHK1/2 and VEGFR2 inhibitors showed varying activity, combinations of TAK228 with either cediranib or AZD7762 showed significant synergism in multiple cell lines. Clinical examination of these combinations agents may prove useful in recurrent endometrial cancer.