Differential Phosphoproteomics Deciphers Physiopathology of High-Risk Mantle Cell Lymphoma

Abstract

Background: Mantle cell lymphoma (MCL) is a rare adult-onset non-Hodgkin lymphoma of which genetic hallmark is CCND1 translocation with IG genes, although CCND2, CCND3 or CCNE translocations are alternatives. The median survival of patients with MCL has lately increased due to improved therapies. However, the identification of high-risk MCLs characterized by pleomorphic or blastoid appearance, high proliferation index and TP53 deletions/mutations, remains mandatory due to their highly aggressive features and dismal clinical course even with intensified regimens. Therefore, specific therapeutic approaches are still warranted for such patients, but clinical and biological data are scarce since high-risk MCLs represent only 10-20% of all MCLs. A valuable source of rare samples for clinical/translational research is archived collections of formalin-fixed paraffin-embedded (FFPE) tissue blocks which enable retrospective studies to be performed. However, FFPE samples remain challenging for omics studies, especially phosphoproteomics. Here we implemented an original process to overcome this technical roadblock and took full advantage of FFPE samples to investigate biological processes differentially involved in high-risk and classical MCL (cMCL) to highlight rationale for innovative targeted therapies for these aggressive forms. Material and methods: We employed an unbiased differential label-free phosphoproteomic approach using archived FFPE MCL samples. Briefly, archived FFPE lymph nodes conclusive for cMCL or blastoid MCL (bMCL) diagnoses were retrieved from the Biological Resource Center of Strasbourg University Hospital from 1996 to 2016. H&E slides were reevaluated, and areas enriched in tumoral cells were selected to be punched from FFPE blocks. Our original sample preparation procedure entailed 1/ xylene-based deparaffinization, 2/ protein extraction with high content SDS-based lysis buffer, 3/ sample reduction/alkylation followed by cleanup and protein digestion on carboxylate paramagnetic beads and Lys-C/trypsin proteolytic enzyme mix, 4/ phosphopeptide enrichment on the AssayMap Bravo platform (Agilent) using IMAC Fe(III)-NTA cartridges. All samples were then analyzed on a NanoElute coupled to a TimsTOF Pro mass spectrometer (Bruker). Label-free quantification was performed using MaxQuant with the human database downloaded from SwissProt in April 2022. Further bioinformatics analyses were conducted in Perseus and Prostar. Data were functionally annotated with Reactome Pathway Database and STRING for association networks. Results: For this monocentric study we retrieved 30 FFPE blocks of lymph node biopsies conclusive for MCL diagnosis, including 15 bMCL and 15 cMCL. Although FFPE samples represented a challenge to work with for phosphoproteomics, we extracted enough protein from three 2mm-punches to proceed to the phosphopeptide enrichment according to our original sample preparation procedure. Overall, we successfully identified and quantified 7,220 class I phosphosites, i.e., sites of phosphorylation for which the probability of the phosphate group localization is greater than 0.75. No significant difference was found considering neither the archived period length of FFPE blocks nor the MCL subtypes (bMCL or cMCL). Differential analysis of bMCL and cMCL identified 180 differentially phosphorylated phosphosites (p-value < 0.05) corresponding to 103 proteins. Among these phosphosites, 139 were more phosphorylated in bMCL. Functional annotation of their corresponding proteins highlighted an over-representation of proteins involved in cell cycle, DNA repair and RNA metabolism within bMCL differential phosphoproteome (Fig.1). In addition, the kinase enrichment analysis of these differentially phosphorylated proteins predicted an increased activity of cyclin-dependent kinases in bMCL compared to cMCL. Conclusion: Here we performed for the first time phosphoproteomics from archived FFPE MCL samples. Differential analysis revealed that high-risk MCLs are characterized by an increased phosphorylation of proteins involved in cell cycle and an increased activity of cyclin-dependent kinases. Further studies are mandatory to fully understand to which extent cyclin-dependent kinases could be suitable targeted therapeutic candidates for patients with high-risk MCL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal