Abstract 2933: Long-term BRAF(V600E) inhibition results in a spontaneous KRAS(G12D) mutation and increased epithelial to mesenchymal transition (EMT) in papillary thyroid cancer cells (PTC)

Abstract

Abstract Objective: Activating mutations in BRAF(V600E) are commonly observed in PTC and result in a functional dependence upon the constitutively active MAPK pathway. While targeted inhibitors are initially effective, inevitably cells develop alternative mechanisms of pathway activation. Mechanisms of primary resistance have been described in thyroid cancer cell lines, however acquired resistance has not. Our study investigates adaptive mechanisms of BRAF(V600E) inhibitor resistance and accompanying metastatic phenotypes in PTC cells following long-term vemurafenib exposure. Materials & Methods: KTC1 sub-cell lines (PTC cells, BRAFV600E) were developed following treatment with either 0.25 or 1.0 μM of vemurafenib, a BRAF V600E inhibitor, for >20 passages. Western blot and qRT-PCR were used to assess EMT marker expression and pathway activation. 2D and 3D invasion assays, immunofluorescence microscopy, and direct cell count growth assays were used to assess inhibitor resistance and metastatic phenotypes. Sequenom was used to detect acquired mutations. Results: A vemurafenib resistant (VR) subline of KTC1 cells was derived following long-term treatment with the drug. Resistance coincided with spontaneous acquisition of a KRAS(G12D) activating mutation. Our data show KRAS(G12D) driven vemurafenib-resistance significantly enhanced expression of distinct EMT markers and translated into increased growth, migration, and invasion, when compared to the control sub-lines. This gain in metastatic phenotype coincided with increase activation of both the AKT and ERK pathways. Conclusion: Our results suggest an acquired KRAS mutation confers BRAF V600E inhibitor resistance and a more aggressive metastatic phenotype in vitro. Our hope is that further study of the mechanisms of resistance will add to potential markers for early detection of BRAF inhibitor resistance, leading to improved patient outcomes. Citation Format: Brian P. Danysh, Maria E. Cabanillas, Marie-Claude Hofmann. Long-term BRAF(V600E) inhibition results in a spontaneous KRAS(G12D) mutation and increased epithelial to mesenchymal transition (EMT) in papillary thyroid cancer cells (PTC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2933.