Abstract 2006: Kinases and adaptive signaling contribute to drug resistance in BRAF mutant melanoma
Authors: Sharma Ritin , Phadke Manali , Britton David , Pike Ian , Smalley Keiran , Koomen John M. 
Published: August 2015

Melanoma, the most lethal form of skin cancer, is marked by numerous genetic modifications, including point mutations as well as overexpression and deletion of genes. Intrinsic and acquired resistance to BRAF V600E targeted therapies (BRAFi) in metastatic melanoma patients further underscores the need for global profiling of melanoma circuitry at the functional level. Therefore, activity-based protein profiling (ABPP) and phosphoproteomics was carried out to decipher steady state differences in global signaling mechanisms in naïve and BRAFi resistant melanoma cell lines with BRAF V600E mutations.

Four cell lines (A375, 1205Lu, WM164 and WM793) were selected to evaluate different molecular backgrounds of BRAF mutation based on their PTEN status (either WT or null). For each cell type, both the naïve and BRAFi resistant lines were analyzed via ABPP as well as chemical labeling with tandem mass tags (TMT) prior to discovery phosphoproteomics. While the ABPP approach mined for kinases, phosphoproteomics identified STY phosphorylated peptides providing information on signaling via kinase substrates. LC-MS/MS discovery proteomics (RSLC and Q Exactive, Thermo) identified and relatively quantified all peptides observed in ABPP and TMT phosphoproteomics experiments. MaxQuant was used for data evaluation; preliminary statistical analyses were performed in Perseus to select significant differences for pathway mapping (GeneGO, Metacore) and follow up experiments using siRNA or pharmacological inhibition. Adaptive responses to combination treatment were also explored using the SysQuant workflow for quantitative expression analysis and phosphoproteomics. These experiments served as a basis for comparison for a pilot project of 12 metastatic tumors from BRAF mutant melanoma patients selected for comparison of good and poor survival outcomes.

ABPP measurements on different cell line models (A375, 1205Lu, WM793 and WM164) reveal significant differences in ATP uptake of proteins in the resistant cell line model compared to its naïve counterpart. For example, in the 1205Lu cell line several proteins including EGFR, p38alpha, DNA-PK formed an interconnected pathway. Overall we identified between 2,000-2,800 proteins in each cell line with ∼150 kinases. Isobaric labeling coupled to phosphoproteomics identified ∼1,600 quantifiable proteins with ∼4,000 phosphorylation sites. Phosphoproteomics revealed concomitant increase in phosphorylation levels of the substrates acted upon by kinases showing higher ATP uptake in ABPP measurements. For example, in 1205Lu BRAFi resistant cells, CDK1, CDK2 and DNA-PK showed higher ATP uptake and their substrates SSK1, DPYSL3, and vimentin showed higher phosphorylation levels.

The complementary nature of the two functional proteomics approaches provided holistic overview of signalling network in melanoma and enabled selection of targets for follow-up studies.

Citation Format: Ritin Sharma, Manali Phadke, David Britton, Ian Pike, Keiran Smalley, John M. Koomen. Kinases and adaptive signaling contribute to drug resistance in BRAF mutant melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2006. doi:10.1158/1538-7445.AM2015-2006