Integrated molecular profiling of melanoma cell lines reveals genotype-drug phenotype associations giving insight on development of combination therapies

Adaptive survival responses to targeted therapy reduce therapeutic benefit, and thus there is a need to identify drug combinations that co-target both oncogenic drivers and critical adaptive responses. We screened a panel of twelve V600E BRAF melanoma cell lines with a library of targeted drugs, and identified the combination of lapatinib (a HER family inhibitor) plus PLX4720 (a RAF inhibitor) as synergistically cytotoxic in the six lines that displayed complete or partial resistance to PLX4720 alone. To understand the mechanism(s) of drug resistance and synergy, and to identify potential biomarkers for utilization of this combination, we have performed an extensive molecular analysis of the cell lines and their responses to treatment with the drugs, singly and in combination. Alamar blue was used to calculate the cytotoxic effects of the drugs, and the cells were profiled by gene expression microarrays and reverse phase protein microarrays, before and after drug treatment. DNA methylation microarrays, and exome sequencing were used to identify the epigenetic and genetic context of the lines. Unsupervised hierarchical clustering analysis of the cytotoxic effects of the single drug and combinatorial treatments revealed four well defined groups: synergistic to combination treatment, PLX4720 sensitive, lapatinib resistant, and insensitive to either drug. The cell lines were analyzed using principal component analysis on the basal gene expression profiles. This analysis revealed four major clusters whose membership was identical to the clusters observed in the cytotoxicity analysis, indicating a strong genotype-drug phenotype relationship (i.e. cell lines that showed similar basal transcriptional programs also demonstrated similar responses to treatment. Differential gene expression analysis was performed between the synergistic group and each of the other groups. The most prominent gene expression differences were between the synergistic and PLX4720 sensitive lines, which correlated with differences in MITF expression and transcriptional output. Changes in gene expression and phosphoproteome levels in response to PLX4720 and lapatinib treatment between the four groups were used to identify genes and pathways associated with drug resistance and synergy. Genes involved in apoptosis and cell cycle regulation were found that could play a role in conferring resistance and synergistic response to PLX4720 and lapatinib treatment. These differing patterns of adaptive responses provide insights into molecular mechanisms of drug resistance and synergy and a pathway to rational construction of drug combinations.

Citation Format: Brian J. Capaldo, Devin G. Roller, Mark Axelrod, Alex Koeppel, Michael J. Weber, Aaron Mackey, Dan Gioeli, Stefan Bekiranov. Integrated molecular profiling of melanoma cell lines reveals genotype-drug phenotype associations giving insight on development of combination therapies. [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 4166. doi:10.1158/1538-7445.AM2014-4166