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Individualized proteogenomics in analysis of resistance to BRAF inhibition in malignant melanoma
Author(s) -
Schmitt Marisa,
Nalpas Nicolas,
Maass Annika,
Macek Boris
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.934.3
Subject(s) - vemurafenib , melanoma , biology , cancer research , stable isotope labeling by amino acids in cell culture , phosphoproteomics , mapk/erk pathway , kinase , protein kinase a , phosphorylation , signal transduction , protein phosphorylation , proteomics , biochemistry , gene , metastatic melanoma
Malignant melanoma is characterized by somatic BRAF and RAS mutations in the mitogen‐activated kinase pathway (MAPK), which strongly correlate with poor prognosis of the disease. The serine‐threonine kinase BRAF is mutated in ~70% of melanoma cases, resulting predominantly in V600E substitution and leading to constitutive activation of downstream MAP kinase signaling pathways. Targeted inhibition of the mutated BRAF with selective inhibitors like vemurafenib or dabrafenib results in the reduction of MAPK signaling and regression of the disease. However, most patients quickly develop resistance to drug treatment. Several mechanisms for the acquired resistance in melanoma have been detected in vivo and in vitro , but key (phospho)proteins involved in resistance as well as somatic mutations that can alter their modification status and can therefore influence development of resistance are still largely elusive. Here we use quantitative proteogenomics based on stable isotope labeling with amino acids in cell culture (SILAC) to investigate the proteome of five drug‐resistant and drug‐sensitive melanoma cell lines. Among 7,808 quantified proteins and 13,452 phosphorylation sites, we detect 53 proteins and 312 phosphorylation sites involved in major biological processes and cancer‐related signaling pathways that were significantly changing in resistant cells and therefore constitute potential therapeutic targets or markers of the resistance to BRAF inhibitors. To validate our data set, we focused on several interesting candidates and validated their levels by western blot across all investigated cell lines. We are currently knocking‐out respective genes using CRISPR/Cas9 and assaying genome‐edited melanoma cell lines for variability, cell migration, cell invasion and cell apoptosis. To study the influence of non‐synonymous somatic mutations on the signal transduction networks, we established a bioinformatics workflow for prediction of non‐synonymous single nucleotide variants (nsSNVs), as well as insertions or deletions (InDels) of nucleotides, and applied it to exome sequencing data from the vemurafenib‐sensitive and resistant melanoma cell line A375. This led to A375‐specific protein database containing 18,786 single amino acid variants (SAVs), of which 1,325 have not been reported previously. Over 8,800 SAVs affected modifiable amino acid residues Ser/Thr/Tyr and Lys, therefore potentially influencing signal transduction networks involved in resistance. To conclude, our (phospho)proteomics data provide important insights into molecular mechanisms underlying resistance to BRAF inhibition and the established proteogenomics workflow can be directly used for a personalized approach to cancer understanding and individualized treatment.