Fig 1.
LY2090314 is a GSK inhibitor which elevated Wnt signaling in melanoma cell lines.
A. Structure of LY2090314. B. Riluzole (▲), LY2090314 (■) and BIA (●) activated the TCF/LEF luminescent reporter in A375 cells following 5 hours of drug exposure. C. LY2090314 (20nM) decreased phosphorylation of GSK3α/β in A375 cells and increased β-catenin and Axin2 protein levels as determined by Western blot. D. LY2090314 (20nM) treatment of A375 cells increased Axin2 gene expression in a time dependent manor. E. Uncomplexed β-catenin IP experiments reveal a large increase in β-catenin protein following LY2090314 (20nM) treatment for 48 hours.
Fig 2.
LY2090314 potently induces apoptotic cell death in a range of melanoma cell lines irrespective of BRAF mutation status.
A. 72hr cytotoxicity assays (CellTiter-Glo) reveal LY2090314 activity in the nanomolar range in numerous melanoma cell lines. The commercially available GSK3 inhibitor BIA demonstrates activity in the micromolar range. B. Cleaved caspase assays (Promega) reveal an induction of cleaved caspase3/7 prior to cell death in melanoma cell lines, data presented is following 72 hours LY2090314 drug treatment. C. Cleaved PARP can be detected prior to cell death following cell treatment with LY2090314 indicating that LY2090314 induces cell death via apoptosis.
Fig 3.
Cell death induced by LY2090314 is dependent on β-catenin and GSK3β knockdown increases the sensitivity of cells to LY2090314.
A. Melanoma cells stably transfected with shRNAs targeting β-catenin display decreased β-catenin and Axin2 protein expression by western blot. A375 (B) and M14 (C) cells expressing shRNAs targeting β-catenin (● Control; ■ β-catenin shRNA 1; ▲ β-catenin shRNA 2; ▼ β-catenin shRNA 3) become resistant to LY2090314 suggesting that β-catenin is required for apoptotic cell death in response to LY2090314. D, E. A375 cells targeted with GSK3β shRNA, but not GSK3α shRNA, demonstrates increased sensitivity to LY2090314 (4.5nM, 72hr).
Fig 4.
LY2090314 demonstrates activity in cell lines resistant to the BRAF inhibitor Vemurafenib and has an independent mechanism of action.
A. Wild type melanoma cell lines M14 and A375 are sensitive to growth inhibition by Vemurafenib relative to cells selected as resistant to Vemurafenib. However, LY2090314 retains potency in both wild type and Vemurafenib resistant cell lines (● A375 control; ■ Vemurafenib resistant A375; ▲ M14 control; ▼ Vemurafenib resistant M14). B. In a panel of melanoma cell lines, variable sensitivity to Vemurafenib can be observed using 72 hr cytotoxicity assays whilst all cells tested displayed sensitivity to LY2090314. C. LY2090314 and Vemurafenib have distinct mechanisms of action. Following drug treatment, cells were analyzed for compound effect on the Wnt and Ras pathways and demonstrate differential signaling pathway modulation.
Fig 5.
LY2090314 elevates Axin2 gene expression in vivo, demonstrates single agent activity in the A375 xenograft model of melanoma and enhances the efficacy of DTIC.
A. Plasma PK of LY2090314 in non-tumor bearing mice. B. A single dose of LY2090314 (25mg/kg) elevates Axin2 gene expresion in vivo, a marker of wnt pathway activation. C. Subcutanous A375 xenografts were treated with 25mg/kg LY2090314 Q3D and resulted in a significant tumor growth delay (p<0.006). D. LY2090314 (2.5mg/kg Q3D) enhances the efficacy of DTIC (60mg/kg QD) in A375 xenografts (p< 0.02).