2017R-Proteomics-Impact of phosphoproteomics in the translation of kinase-targeted therapies

Table 1 Kinase inhibitors approved by the US FDA for the treatment of cancer and other diseases

ALL: Acute lymphoblastic leukaemia; CCRCC: clear cell renal cell carcinoma; CEL Chronic eosinophilic leukaemia; CLL: Chronic lymphocytic leukaemia; CML: Chronic myeloid leukaemia; CRC: Colorectal cancer; DFSP: Dermatofibrosarcoma protuberans; GIST: Gastrointestinal stromal tumour; HCC: Hepatocellular carcinoma; HES: Hypereosinophilic syndrome MDS: Myelodysplastic syndrome; MTC: Medullary thyroid cancer; NSCLC: Non-small cell lung carcinoma; RCC: Renal cell carcinoma; SCC: Squamous cell carcinoma; TSC: Tuberous sclerosis complex. (a) Granted a breakthrough designation by the FDA in February 2016. (b) Flt-3, c-Kit, VEGFRs, EGFR, PDGFR, Syk, Src, PKA, PKCα, PKCβ, PKCδ, PKC (c) No direct binding to mTOR, interacts with the FKBP12 component of the mTORC1 specifically inhibiting mTOR when is part of this complex. (d) Human mAb. (e) Humanized人源化 mAb. (f) Chimeric mAb嵌合单克隆抗体.

Figure 1. Technologies and procedures implemented for the analysis of the phosphoproteome

(A) Strategies developed for the study of the phosphoproteome. Antibody based procedures includes Western blot (WB), immunohistochemistry免疫组织化学 (IHC), ELISA or new multiplex assays多元分析. MS based procedures are targeted or untargeted when specific or as many as possible phosphopeptide ions are tracked. Targeted methodologies include SRM, MRM, SIM or PMR. Acquisition in untargeted techniques are data dependent (DDA) when the peptide ions are randomly selected for fragmentation based on their intensity, the peptides can be unlabelled or chemically or metabolically labelled. Unlabelled peptides are quantified using the number of spectra or extracted ion chromatograms (XIC) of the precursor peptide ions. In data independent acquisition procedures (DIA) like sequential window 连续窗口acquisition of all theoretical mass spectra (SWATH) and MSe, all peptides in a defined m/z are fragmented.
(B) Schematic workflow of an untargeted data dependent phosphoproteomics experiment. Before cell lysis biological specimens样本 can be labelled with SILAC. After an optional fractionation by SDS-PAGE, IP or IEF, proteins are reduced, alkylated 烷基化and digested with trypsin or other enzymes. After optional steps of chemical labelling with iTRAQ or tandem mass tag (TMT) and fractionation with SCX, phosphopeptides are enriched using IMAC, metal oxide affinity chromatography (MOAC) or antibodies against phosphorylated residues. For the acquisition and processing of mass spectrometry data, samples are run in an LC-MS/MS system and ion peaks are linked to peptides and quantified using specialised software. Further computational analysis is done to establish statistically regulated phosphopeptides between experimental conditions. Regulated phosphopeptides are used to determine enrichment of particular pathways or to infer kinase activity using scripts like kinase substrate enrichment analysis (KSEA).

Figure 2. Application of phosphoproteomics studies to obtain translational information linked to kinase inhibitors

Illustrative example showing how phosphoproteomics can answer questions
relating the use of kinase inhibitors. In this example, an AKT and MEK mediated signalling network, in which an activating mutation in a receptor tyrosine kinase激酶 generates a pathological病理 response, is ‘treated’ with the MEK inhibitor trametinib to repress such response. Measuring the phosphoproteome of a pathological tissue allows determining whether the targeted kinase (1) or the pathways that act in parallel of such kinase (5) are active. Profiling phosphoproteome alterations in response to kinase inhibitors can determine if a drug is inhibiting its target kinase (2) or the effectors downstream of this kinase (4). Phosphoproteomics profiling can also identify new substrates that are directly (7) or indirectly downstream of the targeted kinase (8) and thus identify new biological processes linked to the target kinase (9). Profiling the responses to kinase inhibitors can also provide information about off-target effects of the compounds (3) as well as feedback loops released by the inhibition of the targeted kinase (6).