An integrated approach to the study of mantle cell lymphoma

Understanding the mechanisms of disease is the base of modern medicine. Cancer research has become over the years more and more interdisciplinary, using techniques developed initially in other science disciplines, such as chemistry, experimental biology, immunology, mathematics, informatics and many others. In recent years, with the advent of high-throughput techniques, this multi-faceted approach has become a necessity more than a luxury. In this project we exploited an integrated approach to the study of mantle cell lymphoma (MCL). To gain a more complete, and possibly more real, picture of MCL pathogenesis we tried to integrate information coming from proteomic, transcriptomic and genomic studies acquired by means of several modern high-throughput techniques. In the first part of this project we took advantage of a shotgun proteomic technique, called Phoshoscan, to identify the tyrosine-phosphorylation profile of MCL cells. Since tyrosine phosphorylation regulates the activity of many proteins, this approach allowed us to identify the most active pathways in the analyzed samples. The Phosphoscan analysis has shown that MCL bears an aberrant activation of the B-cell receptor (BCR) signaling pathway, and that at least in vitro the inhibition of Syk (a key molecule of BCR signaling) is lethal for MCL cells. Other kinases were also found to be active (phosphorylated) and these are matter of current investigation by means of pharmacological inhibition. However, the mechanism driving BCR activation remained elusive. Since the presence of activating mutations is one basic mechanism of cancer pathogenesis, we hypothesized that a genetic lesion in one or more of the active proteins might be responsible for the abnormal activation of the BCR pathway. To test this hypothesis we looked for the presence of mutations in the coding sequences corresponding to the most abundant Tyr-phosphorilated proteins, taking advantage of the classical Sanger sequencing. We identified four missense mutations in PRPF4B, BTK, PRKCD and CD79B genes but further functional studies and an extensive validation are necessary to assess these mutations role. Since BCR stereotypy and activation have been linked to the pathogenesis of different lymphomas and leukemias, like B-CLL, we also investigated whether MCL cell lines bore stereotyped BCR, as an additional mechanism stimulating the signaling cascade. We determined the precise sequence of rearranged heavy and light chain genes in several MCL cell lines and we found no evidence of heavy chain stereotypy, but recurrent presence of stereotyped light chain. These findings highlight the possible role of light chains in tonic BCR signaling and provide the basis to use MCL cell lines as a model to investigate the role of specific BCRs in the pathogenesis of MCL. In the second part of the work we applied a next-generation sequencing (NGS) technique, RNA-sequencing, to seven cases of MCL and three non-neoplastic lymph node samples. This whole-transcriptome approach has the advantage to provide, beyond a complete mutational profile of the expressed genes, several new data (quantitative gene expression profiling, usage of splice variants, chimeric transcripts etc) which can be useful to better understand the molecular pathogenesis of MCL. A preliminary supervised analysis of RNA-seq data identified eleven putative relevant single-nucleotide variants (SNVs), already annotated in the COSMIC database. Only four of them, affecting NOTCH2, ATM, GOT2, and CHPF2 genes, were validated. Moreover we showed that although fusion transcripts are usually present in MCL, they are detectable in a very limited number of cases (i.e. they are mostly private) and do not produce any detectable protein. However the information obtained from this analysis is extremely wide and complex and deserves a deeper investigation by means of bioinformatic analysis and biological validation that is still ongoing. Overall, these analyses brought us new insights about the mechanism driving the pathogenetic process in MCL, highlighting the role of BCR signaling pathway.