Global Phosphoproteomic Analysis of B-Cell Lymphoma Signatures Reveals the Role of Active B-Cell Receptor Signaling in Germinal Center Derived NHLs

Reversible phosphorylation is a fundamental post-translational modification which regulates cellular functions by modulation of the activity of critical proteins in cellular signal transduction pathways. Deregulation of protein phosphorylation underlies the pathogenesis of many human diseases, especially cancers. Large scale phosphoproteomic analysis offer an opportunity to catalogue and understand changes in protein phosphorylation associated with cancers and thus provide new possibilities for discovering novel diagnostic biomarkers and new therapeutic targets. Although B-cell non-Hodgkin lymphomas (NHLs) represent the seventh most common cancer in western countries, the extent to which phosphoprotein deregulation may be involved in their pathogenesis is largely unknown. Here we report results of a label-free quantitative phosphoproteomics study of 3 B-cell NHL entities and investigate the contribution of one of the identified protein in the germinal center-derived NHL proliferation.

Six milligrams of protein from 13 human B-NHL-derived cell lines (4 mantle cell lymphomas (MCL), 3 Burkitt lymphomas (BL) and 6 follicular lymphomas (FL)) digested by trypsin were subjected to phosphopeptide enrichment using metal oxide affinity chromatography (MOAC) and immunoprecipitation using a cocktail of 3 anti-phosphotyrosine antibodies. Phosphopeptides were subjected to liquid chromatography (LC) and MS/MS. Spectra were searched against the UniProtKB database using X!Tandem with k-score. Search results were then post-processed with PeptideProphet and ProteinProphet. All proteins at false discovery rate of 1% were considered for further analysis. Quantitation of identified peptides was based on spectral counts of phosphorylated peptides. Immunoprecipitation and western blot studies were performed to validate the differential phosphorylation of a subset of proteins. The functional consequences of perturbation of an outlier tyrosine phosphorylated protein (PAG1) were explored using shRNA-mediated depletion followed by both colony formation and proliferation assays with or without BCR stimulation by anti-IgM or lipopolysaccharide (LPS).

Quantitative phosphoproteomics revealed a total of 7326 serine/threonine phosphorylatided peptides and 392 tyrosine phosphorylated peptides corresponding to 420, 290 and 356 phosphorylated proteins in BL, FL and MCL, respectively. While a subset of phosphoproteins was identified across all lymphoma entities, hierarchical clustering analysis revealed distinctive signatures for all 3 NHL subtypes. Regarding proteins identified with phosphorylated tyrosine residues, BL and FL cell lines were characterized by phosphorylation of proteins implicated in active B-cell receptor signaling such as BTK, LYN, LCK and SYK whereas MCL cell lines were characterized by phosphorylation of proteins implicated in cell cycle control such as CDK1 and CDK2. Interestingly, we identified PAG1, a negative regulator of B-cell receptor signaling, as the most highly tyrosine phosphorylated protein in the germinal center derived cell lines (BL and FL) exclusively. Differential phosphorylation of several proteins was validated by immunoprecipitation and western blot studies. PAG1 silencing in BJAB (BL cell line) by a specific PAG1 shRNA resulted in an increased propensity for colony formation when compared to a scramble shRNA. PAG1 depletion also enhanced significantly the proliferation of BJAB 48h after BCR stimulation by either anti-IgM (3.9-fold increase versus 3-fold increase, p = 0.016) or LPS (4.2-fold increase versus 3-fold increase, p = 0.015).

Our study reveals the utility of unbiased phosphoproteome interrogation of B-NHLs to characterize signaling networks that may provide insights into the pathogenetic mechanisms. We identify more than 500 phosphorylated proteins in each NHL entity revealing distinct protein phosphorylation signatures between MCL and germinal center derived lymphomas. Activation of the BCR signaling pathway is characteristic of and pathologically relevant in germinal center derived NHLs. The results of our study reveal novel phosphorylation sites and signatures that may be exploited as diagnostic biomarkers or therapeutic targets.

No relevant conflicts of interest to declare.