Integration of Individualised Proteogenomics Datasets to Analyse Single Amino Acid Variants in Cancer

Analysis of patient-specific single nucleotide variants, genomic insertions, deletions and structural variants is a cornerstone of personalised medicine. Although only about 2% of the genomic sequence is protein-coding, mutations occurring in these regions have potential to influence protein structure and can therefore have severe impact on the aetiology of many diseases. Of special interest are mutations that affect modifiable amino acid residues, as protein modifications involved in signal transduction networks cannot be analysed by genomics. Proteogenomics addresses this impact by analysing proteomes in context of patient- or tissue-specific non-synonymous single nucleotide variants (nsSNVs), insertions and deletions. Here we present a bioinformatics application termed Proteogenomics Characterisation Tools (PCTi) that enables straightforward integration of nucleotide variants into protein databases, assessment of their potential impact in different biological context and subsequent visualisation of proteogenomics data. We used PCTi to compare the non-synonymous nucleotide variant landscape, transcriptome expression and (phospho)proteome abundance in context of resistance to BRAF inhibitor vemurafenib within the malignant melanoma cell line A375. Based on this, PCTi allowed the reconstruction of the signalling network associated with BRAFi resistance in A375. Subsequently, PCTi was able to prioritise key actionable nodes from this network and predict drug therapies to disrupt BRAFi resistance mechanism in A375; such as the key hub AURKA that can be inhibited using the drug Alisertib. Furthermore, we investigated nucleotide variants that interfere with the protein modification status and potentially influence signal transduction networks. MS measurements confirmed mutation-driven modification changes in approximately 50 proteins; among these was the transcription factor RUNX1 mutated on S276L. We confirmed the loss of the Ser276 phosphorylation site by MS and demonstrated the impact of this mutation on RUNX1 interactome.