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.
Highlights
Accumulation of mutations is one of the hallmarks of cancer cells and malignant melanoma is a type of cancer with the highest frequency of somatic mutations (1)
These multi-omic measurements were integrated (1) to reconstruct signalling networks that are disturbed in BRAFi-resistant A375 cells; and (2) to investigate the impact of variants altering protein post-translational modification (PTM) status in A375 cells irrespective of BRAFi resistance
Variance in protein abundance discriminates between BRAFi-sensitive and -resistant A375 cells Initially, we investigated the mutational landscape of A375 R and A375 S cells by high-throughput sequencing
Summary
Accumulation of mutations is one of the hallmarks of cancer cells and malignant melanoma is a type of cancer with the highest frequency of somatic mutations (1). The predominant BRAF mutation is within the kinase domain with a single nucleotide substitution of valine to glutamic acid at amino acid 600 (4). This mutation can result in a 500-fold increased, dimerization-independent activation of BRAF, and leads to a constitutive activation of downstream signalling in cancer cells (3, 5). Targeted inhibition of the mutated BRAF kinase with selective inhibitors like vemurafenib, dabrafenib or encorafenib (BRAFi) results in a reduction of MAPK pathway signalling (5). The identification of multiple cellular mechanisms of resistance has greatly improved the understanding of malignancy and clinical outcomes of BRAFV600E metastatic melanoma e.g. by the introduction of combined BRAF and MEK inhibition
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