Abstract 3381: Overcoming resistance to BRAF inhibitors in colon cancer and melanoma cell lines.
Authors: Kelter Gerhard , Krumbach Rebekka , Giesemann Torsten , Vuaroqueaux Vincent , Metz Thomas , Metcalfe Thomas , Fiebig Heinz-Herbert 
Published: August 2014

Mutations in the B-raf oncogene constitutively activate the RAF-MEK-ERK signaling pathway, a key regulator of cell proliferation and survival. The most common BRAF mutation (V600E) occurs in approximately 70% of primary melanomas and 10% of colorectal cancers. Melanoma patients carrying the BRAF(V600) mutation respond with frequency of 80% to the BRAF inhibitor vemurafenib, while colon cancer patients respond at a rate of 5% only, indicative of intrinsic resistance. Moreover, clinical benefit is further limited by the rapid emergence of acquired resistance among vemurafenib responders. Combination therapy targeting both BRAF(V600E) and possible escape pathways appears to be a promising approach to reverse resistance to BRAF inhibitors. Here, we present the molecular characterization and in vitro chemosensitivity profiles of 20 melanoma and 20 human colorectal cancer cell lines. Four of the colon and 11 of the melanoma cell lines were established at Oncotest from patient-derived tumor xenografts (PDX). Molecular profiles include the mutation status of BRAF, PIK3CA, KRAS, and NRAS (exon sequencing) and protein levels for components of the MAPK, EGFR and PI3K/AKT pathways (Western blot analysis). Sensitivity profiles for inhibitors of BRAF, MEK, EGFR and PI3K were determined in vitro using a fluorescence-based survival and proliferation assay. Overcoming resistance to BRAF inhibitors was investigated by combination of vemurafenib with other inhibitors of the RAF-MEK-ERK pathway or inhibitors of potential escape pathways like EGFR, PI3K/AKT or c-MET and evaluated by combination index according to Chou-Talalay.

In line with the clinical situation, the BRAF V600E substitution was found in 13/20 (65%) of the melanoma and only in 4/20 (20%) of the colon cancer cell lines. BRAF and NRAS mutations were mutually exclusive in melanoma cell lines and KRAS mutations did not occur at all. Among the colon cancer cell lines, in addition to the BRAF(v600E) mutation, alteration in KRAS (10/20 cell lines), PIK3CA (6/20) and NRAS (1/20) were detected. Chemosensitivity profiling revealed intrinsic resistance to BRAF inhibitors for 3/13 (23%) BRAF(V600E) melanoma cell lines and for 3/4 (75%) BRAF(V600E) colon cancer cell lines. Reversal of the resistance of BRAF inhibitor-insensitive melanoma cell lines was recorded as synergism between vemurafenib and inhibitors of vasious kinases (c-MET, AKT, PI3K, MEK, or EGFR. A particularly strong synergistic interaction was found for vemurafenib and the c-MET inhibitor SU11274, irrespective of BRAF status. For colon cancer cell lines, the strongest synergisms were detected for the combinations of vemurafenib with inhibitors of c-MET, EGFR, PI3K, AKT and MEK.

Displaying similar mutation and chemosensitivity profiles as melanomas and colon cancers in the clinic, the present cell line panels represent valuable tools to investigate the reversal of resistance towards BRAF inhibitors.

Citation Format: Gerhard Kelter, Rebekka Krumbach, Torsten Giesemann, Vincent Vuaroqueaux, Thomas Metz, Thomas Metcalfe, Heinz-Herbert Fiebig. Overcoming resistance to BRAF inhibitors in colon cancer and melanoma cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3381. doi:10.1158/1538-7445.AM2013-3381