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Proteogenomics Reveals Perturbed Signaling Networks in Malignant Melanoma Cells Resistant to BRAF Inhibition
- Source :
- Molecular & Cellular Proteomics : MCP
- Publication Year :
- 2021
- Publisher :
- American Society for Biochemistry and Molecular Biology, 2021.
-
Abstract
- Analysis of nucleotide variants is a cornerstone of cancer medicine. Although only 2% of the genomic sequence is protein coding, mutations occurring in these regions have the potential to influence protein structure or modification status and may have severe impact on disease aetiology. Proteogenomics enables the analysis of sample-specific nonsynonymous nucleotide variants with regard to their effect at the proteome and phosphoproteome levels. Here, we developed a proof-of-concept proteogenomics workflow and applied it to the malignant melanoma cell line A375. Initially, we studied the resistance to serine/threonine-protein kinase B-raf (BRAF) inhibitor (BRAFi) vemurafenib in A375 cells. This allowed identification of several oncogenic nonsynonymous nucleotide variants, including a gain-of-function variant on aurora kinase A (AURKA) at F31I. We also detected significant changes in abundance among (phospho)proteins, which led to reactivation of the MAPK signaling pathway in BRAFi-resistant A375 cells. Upon reconstruction of the multiomic integrated signaling networks, we predicted drug therapies with the potential to disrupt BRAFi resistance mechanism in A375 cells. Notably, we showed that AURKA inhibition is effective and specific against BRAFi-resistant A375 cells. Subsequently, we investigated amino acid variants that interfere with protein posttranslational modification (PTM) status and potentially influence A375 cell signaling irrespective of BRAFi resistance. Mass spectrometry (MS) measurements confirmed variant-driven PTM changes in 12 proteins. Among them was the runt-related transcription factor 1 (RUNX1) displaying a variant on a known phosphorylation site S(Ph)276L. We confirmed the loss of phosphorylation site by MS and demonstrated the impact of this variant on RUNX1 interactome.<br />Graphical Abstract<br />Highlights • Proteogenomics workflow to study the malignant melanoma cell line A375. • Key signaling pathways are perturbed in BRAFi-sensitive and -resistant A375. • The signaling network in BRAFi-resistant cells can be targeted by several drugs. • Multiple amino acid variants directly affect protein phosphorylation status. • Loss of a phosphorylation site on RUNX1 alters its interactome.<br />In Brief Proteogenomics is a powerful tool to study the mode of action of disease-associated mutations at the genome, transcriptome, proteome, and PTM level. Here, we applied a proteogenomics workflow to study the malignant melanoma cell line A375. Such workflow, used here as a proof of concept on A375 cells, may be applicable to other cancer types, cell lines, or even patient-derived samples.
- Subjects :
- NOG, N-ocetylglucoside
Biochemistry
Interactome
WES, whole-exome sequencing
Analytical Chemistry
AURKA, aurora kinase A
ERK, extracellular signal-regulated kinase
A375 S, BRAFi-sensitive A375 cells
A375 R, BRAFi-resistant A375 cells
Vemurafenib
nucleotide sequencing
mass spectrometry
Proteogenomics
Kinase
phosphorylation
AGC, automatic gain control
LC-MS/MS, liquid chromatography–tandem mass spectrometry
Proteome
Core Binding Factor Alpha 2 Subunit
Phosphorylation
RT, room temperature
medicine.drug
RUNX1, runt-related transcription factor 1
Signal Transduction
Proto-Oncogene Proteins B-raf
FDR, false discovery rate
Biology
Serine/Threonine-Protein Kinase B-Raf
FBS, fetal bovine serum
DHB, dihydroxybenzoic acid
Cell Line, Tumor
medicine
melanoma
cancer
Humans
Molecular Biology
Protein Kinase Inhibitors
SNV, single nucleotide variant
Research
AURKAi, AURKA inhibition
BRAFi resistance
BRAF, serine/threonine-protein kinase B-raf
BRAFi, BRAF inhibitor
MS, mass spectrometry
Drug Resistance, Neoplasm
HCD, higher-energy collisional dissociation
Cancer research
Aurora Kinase A
PTM, posttranslational modification
MAPK, mitogen-activated protein kinase
Subjects
Details
- Language :
- English
- ISSN :
- 15359484 and 15359476
- Volume :
- 20
- Database :
- OpenAIRE
- Journal :
- Molecular & Cellular Proteomics : MCP
- Accession number :
- edsair.doi.dedup.....e5d55738d47a8fb0d6432cd9bffb21f1