Abstract 3699: Molecular profiling of BRAFi-resistance in melanoma cancer models using high-throughput sequencing in patient-derived xenografts
Authors: Zeitouni Bruno , Kelter Gerhard , Maier Armin , Kiefer Florian , Foucault Frederic , Peille Anne-Lise , Kees Tim , Giesemann Torsten , Vuaroqueaux Vincent , Metcalfe Thomas , Fiebig Heinz-Herbert 
Published: May 2015

Patients with metastatic V600E mutant melanomas treated with BRAF inhibitors (BRAFi) frequently develop resistant tumors with aggressive phenotypes. Furthermore, approximately 20-40% of V600E mutant tumors are intrinsically resistant to BRAFi. Suitable models for studying mechanisms of acquired and intrinsic resistance to BRAFi are therefore necessary. In the present study, we applied deep sequencing techniques to identify possible mechanisms of intrinsic and acquired resistance in our collection of melanoma patient-derived xenograft models (MEXFs) treated with BRAFi.

Mutational and expression profiles in MEXFs were characterized by whole-exome sequencing (WES) and HG-U133 Plus 2.0 Affymetrix chips and correlated with BRAFi efficacy data from 3D Tumor Clonogenic Assays (TCA). In addition, four resistant cell lines were created by continuously treating 2D monolayer cultures of tumor cells with Vemurafenib, all of which were initially sensitive to BRAFi and carry the BRAF V600E mutation. Expression profiles and mutations of these cell lines were analyzed from RNA-seq data. Potential gene candidates responsible for conferring resistance were further investigated by Q-PCR and Western-blot experiments.

WES data revealed that the number of mutations per model was highly variable. Some MEXFs showed a hyper-mutated profile (&gt;2000 mutations) and were characterized by specific mutational signatures in agreement with those found in melanoma (Alexandrov et al, Nature, 2013). Mutation frequencies of genes typically mutated in melanoma are very similar to those found in The Cancer Genome Atlas. We observed a high correlation of mutations between our MEXFs and their respective cell lines. Among the models with a V600E mutation, only one (MEXF 462) showed resistance to BRAFi-treatment as identified by 3D TCA analyses. In this model, we identified gene point mutations and a high over-expression of EGFR, MEK1, PDGFRA and NF1 in contrast to the other models, suggesting a specific regulation of the RTK signaling pathways. In 2D assays, synergistic interaction of Vemurafenib and Erlotinib was shown. RNA-seq analysis of the cell line established from MEXF 276, in which resistance to Vemurafenib was induced, revealed around 20% of genes being differentially expressed (FC&gt;2) between sensitive and resistant cell lines. An up-regulation of EGFR was also found in this resistant cell line and was confirmed by Western-blot. In addition, an overexpression of PLAU, a biomarker of invasiveness, was identified. We currently perform expression profiling of three other models resistant to BRAFi. Preliminary results suggest different patterns of gene regulation involved in acquisition of resistance. A precise map of transcriptomic and mutational profiles of the four cell lines will be generated and we are investigating if invasiveness is increased upon acquisition of resistance.

Citation Format: Bruno Zeitouni, Gerhard Kelter, Armin Maier, Florian Kiefer, Frederic Foucault, Anne-Lise Peille, Tim Kees, Torsten Giesemann, Vincent Vuaroqueaux, Thomas Metcalfe, Heinz-Herbert Fiebig. Molecular profiling of BRAFi-resistance in melanoma cancer models using high-throughput sequencing in patient-derived xenografts. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3699. doi:10.1158/1538-7445.AM2014-3699